In Vitro Protein Expression Guide PDF

Bridging the Gap Between Genomics and Proteomics: In Vitro Expression Tools for
Functional Genomics
In Vitro Expression tools are being used in functional genomic approaches as a bridge
between traditional genomics and proteomics.
Paradigms are shifting. The pace at which biotechnology research is being performed has
changed. What once took a decade can now be done in days. Innovative approaches
combine old technologies with new methods, applications and high throughput innovations.
The biotechnology industry and academia are shifting resources from obtaining sequence
information to evaluating gene expression and determining the subsequent protein function.
The biological research focus is moving toward a systematic characterization of genes and
their encoded proteins from the perspective of their expression and function in the living
cellular context. Currently, however, biological scientists require multiple approaches to
determine the many levels of function, including: predictive in silico bioinformatics,
expression proling, extract-based, cell-based and whole animal assays.
Function can be determined at various levels: simple structural characteristics
(the gene/protein sequence, including alternative spliced forms), expression levels
(e.g., mRNA expression levels), modications (e.g., post-translational glycosylation,
phosphorylation, etc.), interactions (e.g., protein:protein) to actual structural or
enzymatic function.
Another paradigm shift is from characterizing single genes or proteins to studying whole
genomes or proteomes or specic pathways in single experiments. Whole genomes,
proteomes or expression proles can be compared between organisms or in different
disease states.
In Vitro Expression technologies offer signicant time savings over cellular and whole animal
approaches and are generally quite easy to perform. This guide outlines many approaches
that have traditionally been used to study aspects of gene product function (Chapters 1, 2, 3,
9 and 10 of this Guide). These approaches as well as newer in vitro applications for
diagnostics (Chapter 4), high throughput screening (Chapter 5) and functional genomics
approaches (Chapters 6, 7 and 8) are based primarily on the coupled eukaryotic systems
(TNT
Systems). Introduced in 1992 and continually improved, the TNT
Systems allow
researchers to rapidly express proteins directly from DNA, including PCR generated
molecules. Proteins can be expressed from total mRNA populations and cloned using
functional screens as cDNAs (Chapter 6, In Vitro Expression Cloning) or detected non-
isotopically using uorescence (Chapter 5). Although the process of coupled
transcription/translation is complex, the use of rapid in vitro expression systems for many
different applications is simple and convenient. The ability to manipulate both the reaction
and the end products of in vitro expression systems will generate even more useful and novel
tools/applications in the future. An intriguing possibility is to consider in vitro expression of
mRNA (as cDNA) as the amplication technology for Proteomics.
Greg Beckler
Promega R&D Proteomics Platform Manager
Molecular Macromolecular Complex Pathway Cellular
Tissue Developmental Organism Healthy vs. Disease State
In Vitro
Expression
Guide
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C O N T E N T S
Chapter Title Page
1 Overview of In Vitro Expression ................................................................................................................... 1
2 Protein-Protein Interactions .......................................................................................................................... 7
3 Protein-Nucleic Acid Interactions .............................................................................................................. 13
4 The Protein Truncation Test ......................................................................................................................... 18
5 Screening Applications ................................................................................................................................. 23
6 Ribosome Display .......................................................................................................................................... 29
7 Large-Scale Protein Synthesis ................................................................................................................... 34
8 Post-Translational Modications ............................................................................................................... 39
9 Protein Function .............................................................................................................................................. 45
P R O M E G A I N V I T R O R E S O U R C E
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(a)
U.S. Pat. Nos. 5,283,179, 5,641,641, 5,650,289, Australian Pat. No. 649289 and European Pat. No. 0 553 234 have been
issued to Promega Corporation for a rey luciferase assay method, which affords greater light output with improved kinetics as
compared to the conventional assay. Certain applications of this product may require licenses from others.
(b)
The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and
5,700,673.
(c)
U.S. Pat. Nos. 4,966,964, 5,019,556 and 5,266,687, which claim vectors encoding a portion of human placental ribonuclease
inhibitor, are exclusively licensed to Promega Corporation.
(d)
U.S. Pat. No. 5,552,302, European Pat. No. 0 422 217 and Australian Pat. No. 646803 have been issued to Promega
Corporation for the methods and compositions for production of human recombinant placental ribonuclease inhibitor.
(e)
U.S. Pat. Nos. 5,324,637, 5,492,817 and 5,665,563, European Pat. No. 0 566 714 B1, Australian Pat. No. 660329 and
Japanese Pat. No. 2904583 have been issued to Promega Corporation for coupled transcription/translation systems that use
RNA polymerases and eukaryotic lysates.
(f)
The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support
the unauthorized or unlicensed use of the PCR process.
(g)
U.S. Pat. No. 5,780,270 has been issued to Promega Corporation for site-directed mutagenesis.
(h)
For research purposes only. Not for diagnostic or therapeutic use. For bulk purchases of this product, contact TosoHaas, 156
Keystone Drive, Montgomeryville, PA 18936, 1-800-366-4875 or 215-283-5000.
(i)
U.S. Pat. No. 5,744,320 has been issued to Promega Corporation for quenching reagents and assays for enzyme-mediated
luminescence.
(j)
U.S. Pat. No. 6,066,462 has been issued to Promega Corporation for quantitation of protein kinase activity.
(k)
The method of in vitro expression cloning is covered by U.S. Pat. No. 5,654,150 assigned to the President and Fellows of
Harvard College.
(l)
For research purposes only. Not for diagnostic or therapeutic use. For nonresearch users of the Tetralink
TM
Resin, contact
TosoHaas, 156 Keystone Drive, Montgomeryville, PA 18936, 1-800-366-4875 or 215-283-5000.
(m)
The RiboMax
TM
LargeScale RNA Production Systems- T7 and T3 (Cat.# P1290 and P1300) are covered by U.S. Pat. No.
5,256,555 and are sold under a license from Ambion, Inc. They are intended for research use only. Parties wishing to use these
products for other applications should contact Ambion, Inc.
(n)
Licensed under one or both of U.S. Pat. No. 5,487,993 and European Pat. No. 0 550 693.
FluoroTect, GeneEditor, pBestluc, SoftLink Tetralink and Transcend are trademarks of Promega Corporation. Dual-Luciferase,
pGEM, SAM
2
, Stop & Glo and TNT are trademarks of Promega Corporation and are registered with the U.S. Patent and
Trademark Ofce.
Bio-Rad is a registered trademark of Bio-Rad Laboratories, Inc. BODIPY is a registered trademark of Molecular Probes, Inc.
Coomassie is a registered trademark of Imperial Chemical Industries. DispoDialyzer and Spectrum are registered trademarks
of Spectrum Laboratories, Inc. Nonidet is a registered trademark of Shell International Petroleum Company, Ltd.Novex is a
registered trademark of Novel Experimental Technology. Sepharose is a registered trademark of Amersham Pharmacia
Biotech Ltd. TriReagent is a registered trademark of Molecular Research Center, Inc. Triton is a registered trademark of Union
Carbide Chemicals and Plastics Co., Inc.
O V E R V I E W O F
I N V I T R O
E X P R E S S I O N
1
C H A P T E R
A b o u t t h e I ma g e :
In this illustration of
transcription and translation,
the DNA double helix
separates at the RNA
polymerase molecule with an
mRNA strand extending
downward from the RNA
polymerase. Towards the 5-
end of the mRNA the two
subunits of a ribosome
assemble to translate the RNA
message into a polypeptide
chain, which will fold into a
protein molecule. Throughout
this In Vitro Expression Guide
various and recent applica-
tions of translation and protein
expression are discussed.
1
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Chapter One: Overview
Contents Page
Introduction .............................................................................................................. 2
Applications:
Genetic Verication and Detection ................................................................................ 3
Functional Analyses ................................................................................................ 3
Molecular Interaction Detection .................................................................................. 3
Molecular Structure and Localization Analyses ................................................................ 4
Molecular Diagnostics .............................................................................................. 4
High-Throughput Screening ........................................................................................ 5
Functional Genomics ................................................................................................ 5
Preparative Synthesis .............................................................................................. 6
Guide Organization .................................................................................................. 6
Introduction
The use of cell-free systems for the in vitro expression of proteins is a rapidly growing area, with appli-
cations in basic research, molecular diagnostics and high-throughput screening. In vitro expression
encompasses two general strategies. The rst is to use isolated RNA synthesized in vivo or in vitro as
a template for the translation reaction (e.g., using Promegas Rabbit Reticulocyte Lysate
(a,b,c)
(Cat.#
L4151) or Wheat Germ Extract (Cat.# L4380) Systems). The second is to use a coupled
transcription/translation system in which DNA is used as a template (e.g., Promegas
TNT
(a,b,c,d,e)
and E. coli S30 Extract
(a,b)
Systems). This DNA may be either a gene cloned into a
plasmid vector (cDNA) or a PCR
(f)
-generated template.
In vitro expression is a rapidly growing and constantly evolving eld. This guide is intended to provide
a general overview of the technology as presented in recent scientic publications.
We wish to thank Wiley-Liss, a
subsidiary of John Wiley & Sons,
Inc., for their permission to
reproduce much of the material in
the chapter by Jagus, R. and
Beckler, G. (1998) Overview of
eukaryotic in vitro translation and
expression systems, Current
Protocols in Cell Biology,
11.1.111.1.13, in this guide.
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O V E R V I E W O F
I N V I T R O
E X P R E S S I O N
C H A P T E R O N E
References
1. Roberts, B.E. and Paterson,
B.M. (1973) Proc. Natl. Acad.
Sci. USA 70, 2330.
2. Melton, D.A. et al. (1984) Nucl.
Acids Res. 12, 7035.
3. Warner, D.R., Basi, N.S. and
Rebois, R.V. (1995) Anal.
Biochem. 232, 31.
4. Pancharatnam, J. et al. (1996)
Molecular Zoology, Ferraris,
J.D. and Palumbi, S.R., eds.,
Wiley-Liss, New York.
Applications
Genetic Verication and Detection
Open Reading Frame (ORF) Expression. Probably
still the most common use of in vitro expression
is simply to decode a nucleic acid to determine
if a gene or ORF is present. In DNA, an ORF
consists of an initiation codon (usually ATG, but
can also be GTG), followed by a sequence of
nucleotides coding for amino acids and ending
with a termination codon (TAA, TAG or TGA).
For example, a prokaryotic DNA (or eukaryotic
cDNA) clone can be analyzed for the presence
of ORFs using an in vitro expression system.
Alternatively, in vitro expression can be used to
verify an ORF predicted by DNA sequencing. In
each case, the resulting proteins are charac-
terized, and the protein size and structure are
correlated to the size and sequence of the gene.
Specic uses include analysis of viral RNA
genomes in which eukaryotic translation
systems are used to determine the number and
function of the viral genes (1), and study of
differential protein expression using total cellular
mRNA or polysomes from different tissues.
Cloned cDNA Expression. Cloned cDNAs
positioned behind a phage promoter (e.g., T7,
T3 or SP6) are commonly used for generating
gene-specic mRNA used to program trans-
lation reactions (2). Run-off 5-capped or
uncapped mRNA can be produced in vitro and
added to translation extracts. Alternatively, these
DNA constructs can be used directly in
eukaryotic coupled transcription/translation
reactions.
Functional Analyses
Enzymatic Activity Analysis. Many proteins
expressed using in vitro systems are correctly
folded and processed and display normal in
vivo enzymatic activity. If the extract system itself
lacks (or has low levels of) the enzymatic activity
of the expressed protein, the resulting translation
reaction can be assayed directly without protein
purication. Another advantage of in vitro
expression is the ability to add exogenous
factors to study enzymatic activity, potentially
eliminating the need for transfection studies.
In one example, expression of adenylate
cyclase (ACIV; 110kDa) in a TNT
System
produced a protein with the same specic
enzymatic activity as ACIV produced from a
baculovirus expression system (3). In another
example, aromatase produced in a TNT
System
reaction supplemented with canine pancreatic
microsomes and recombinant cytochrome P
450
reductase resulted in an active enzyme (4).
Mutation Analysis. Upon cloning a gene, a
number of studies are undertaken to discern the
function of a gene product. A rst step is often
the introduction of a mutation into the gene to
examine the effect on the expressed protein.
Methods of mutagenesis include: i) serial
deletion mutation analysis by progressive
truncation of the 5- or 3-ends of the gene or by
using Bal 31 digestion from an internal single-
cut restriction site; and ii) site-directed point
mutation analysis (e.g., using Promegas
GeneEditor System
(g)
). Both methods can be
used to identify functionally active domains or
residues.
Post-Translational Modication Analysis. Post-
translational modication of the protein, such as
proteolytic cleavage or the addition of sugars,
lipids, phosphate or adenyl groups, is often
required for functional activity. Each in vitro
expression system has its own endogenous
post-translational modication activities. For
example, various phosphorylation, adenylation,
myristoylation, farnesylation, isoprenylation and
proteolytic activities have been observed using
rabbit reticulocyte lysate. Addition of microsomal
membranes allows the study of glycosylation,
methylation and removal of signal sequences.
Because not all the differences between the
various in vitro expression systems are known, it
may be desirable to try both reticulocyte lysate
and wheat germ extract (and in some cases,
E. coli S30 extract) to determine which system
can produce a functional gene product with the
correct post-translational modications. In
addition, cellular extracts or different microsomal
membrane sources (e.g., Xenopus egg
extracts) can be added to provide additional
modifying activities.
Molecular Interaction Detection
Protein-Protein. Specic protein-protein interac-
tions can be detected using in vitro expression
methods. These interactions may include
specic binding (such as antibody-antigen and
ligand-receptor binding), macromolecular
assembly, and formation of functional tran-
scription complexes. In a common application,
one protein partner is expressed in large
amounts and puried from E. coli as a fusion
protein. The other partner is expressed in an in
vitro expression system as a labeled protein and
used as a probe for detection of the interaction.
Often, this technique is used to verify results
from yeast two-hybrid experiments. A variety of
biochemical analysis methods may be used to
characterize the expressed proteins.
Protein-DNA and Protein-RNA. Putative DNA
binding proteins, such as transcription factors,
can be analyzed for their ability to bind to
specic sequences on radiolabeled oligonu-
cleotides. The binding is detected by an
electrophoretic mobility shift assay (EMSA) in
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using rey luciferase as a model protein
(16,17).
Real-Time Translation/Folding Assays. A novel
approach has been developed using a wheat
germ system in which the components for the
luciferase enzymatic assay have been added
directly to the translation reaction and monitored
continuously in real time. Luciferase was shown
to be fully folded and enzymatically active
immediately upon release from the ribosome
(18). However, no luciferase activity was
observed while full-length luciferase remained
attached to the ribosome as a peptidyl-tRNA,
probably because the C-terminal portion of the
enzyme is masked by the ribosome or
ribosome-associated proteins. The investigators
demonstrated that the ribosome-bound enzyme
acquires enzymatic activity when its C-terminus
is extended by at least 26 additional amino acid
residues (19). The results demonstrate that the
acquisition of the nal native conformation by a
nascent protein can occur as the protein is
being synthesized and that folding does not
require release of the protein from the ribosome.
Macromolecular Assembly. It is possible to
express numerous gene products in one
coupled transcription/translation reaction to form
functional transcription factor complexes (20) or
viral particles (21) that are identical to those
formed in the host.
Molecular Structure Analysis. Understanding the
function of integral membrane proteins is
currently limited by the difculty of producing
crystals for use in X-ray diffraction studies. A
method has been developed for probing confor-
mational changes in membrane proteins using
Fourier transform infrared-difference (FTIR)
spectroscopy. In this method, natively folded
polypeptides are expressed in vitro with a site-
specic insertion of a single isotopic label
through amber suppression (22). This method
does not disrupt the protein structure as did
earlier site-directed mutagenesis methods and
should be applicable to a wide range of other
proteins, including those involved in enzyme
catalysis, ion transport and signal transduction.
Molecular Diagnostics
Protein Truncation Test. A growing application of
coupled transcription/translation systems has
been for diagnosis of genetic diseases, a DNA
technology-dominated eld. The protein
truncation test (PTT), sometimes referred to as in
vitro synthesized protein truncation (IVSP) assay,
was rst reported in 1993 as a rapid method for
detecting translation-terminating mutations in the
large gene responsible for Duchenne Muscular
Dystrophy (23) and the Adenomatous Polyposis
Coli (APC) gene responsible for a type of
hereditary colon cancer (24). In these and other
which greater retardation of the protein-DNA
complex is observed when compared to
unbound DNA. Usually the labeled DNA is
added directly to the in vitro expression reaction.
Researchers studying transcription factors such
as NF-B often use wheat germ extracts, as they
do not contain endogenous mammalian
transcription factors. A method has been
reported to remove endogenous DNA-binding
proteins from the reticulocyte system prior to the
translation reaction (5).
DNA-RNA and RNA-RNA. Antisense DNA oligonu-
cleotides can be useful for inhibiting expression
at both the transcription and translation level.
TNT
Systems have been used to rapidly screen

oligonucleotides for those that best arrest trans-
lation (6,7).
Molecular Structure and Localization Analyses
Characterization of Membrane Association. In vitro
expression systems have been successfully
used to express integral membrane proteins. For
example, expression of G-protein-coupled
receptors in TNT
Systems supplemented with

canine microsomal membranes results in the
correct folding and insertion of transmembrane
domains using the expressed signal anchor and
stop transfer sequences (8,9)
Non-Natural Amino Acid Incorporation. Using
technology originally developed in 1976,
Johnson revolutionized in vitro translation by
demonstrating that non-natural amino acids
could be inserted into polypeptides using
epsilon-modied, lysine-charged tRNAs (10).
Further extension of this technology led to the
incorporation of photoactivatible crosslinking or
uorescent groups into polypeptides, followed
by monitoring of the molecular environment as
the labeled peptides pass through the ribosome
and enter the endoplasmic reticulum pore
(11,12). Site-directed incorporation of a
photoactivable crosslinker through a non-natural
amino acid incorporation was used to capture
protein interacting with different portions of a
protein of interest (13). Other groups have
developed site-specic methods that utilize an
amber suppressor tRNA charged with any
number of non-natural amino acids, including
uorescent, spin-label and isotopic groups (14).
For a review of tRNA-mediated protein
engineering (TRAMPE) see reference 15.
Protein Folding and Chaperonin Interactions. In vitro
expression is increasingly being used to under-
stand the nature of sequential chaperonin
interactions required for protein folding and
localization. Researchers in this eld have
combined the advantages of in vitro expression
with the power of instantaneous reporter gene
product assays. The folding of polypeptides
emerging from ribosomes has been analyzed
References (continued)
5. Ebel, T. and Sippel, A. (1995)
Nucl. Acids Res. 23, 2076.
6. Milner, N., Mir, K.U., and
Southern , E.M. (1997) Nat.
Biotechnol. 15, 537.
7. Lima, W.F. et al. (1997) J. Biol.
Chem. 272, 626.
8. Bayle, D., Weeks, D. and
Sachs, G. (1997) J. Biol. Chem.
272, 19697.
9. Bayle, D. et al. (1997) J.
Recept. Signal Transduct. Res.
17, 29.
10. Johnson, A.E. et al. (1976)
Biochemistry 15, 569.
11. Do, H. et al. (1996) Cell 85,
369.
12. Hamman, B.D. et al. (1997) Cell
89, 535.
13. Zhao, L. et al. (1999) J. Biol
Chem. 274, 14198.
14. Noren, C.J. et al. (1989)
Science 244, 182.
15. Rothschild, K.J. and Gite, S.
(1999) Curr. Opin. Biotech. 10,
64.
16. Frydman, J. et al. (1994) Nature
370, 111.
17. Frydman, J. and Hartl, F.U.
(1996) Science 272, 1497.
18. Kolb, V.A., Makeyev, E.V. and
Spirin, A.S. (1994) EMBO J. 13,
3631.
19. Makeyev, E.V., Kolb, V.A. and
Spirin, A.S. (1996) FEBS Lett.
378, 166.
20. DiDonato, J.A. and Karin, M.
(1993) Promega Notes 42, 18.
21. Sakalian, M. et al. (1996) J.
Virol. 70, 3706.
22. Sonar, S. et al. (1993)
Biochemistry 32, 13777.
23. Roest, P.A. et al. (1993) Hum.
Mol. Genet. 2, 1719.
5
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C H A P T E R O N E
24. Powell, S.M. et al. (1993) N.
Engl. J. Med. 329, 1982.
25. Hogervorst, F.B. et al. (1995)
Nat. Genet. 10, 208.
26. Jagus, R. and Beckler, G.S.
(1998). Overview of eukaryotic
in vitro translation and
expression systems. Current
Protocols in Cell Biology
11.1.111.1.13. Copyright
1998 by John Wiley & Sons,
Inc. Reproduced by permission
of Wiley-Liss, Inc., a subsidiary
of John Wiley & Sons, Inc.
27. Grentzmann, G. et al. (1998)
RNA 4, 479.
28. Schneider, C. et al. (1996) Proc.
Natl. Acad. Sci. USA 93, 14536.
29. Thulasiraman, V. and Matts,
R.L. (1996) Biochemistry 35,
13443.
30. Kuiper, G.G. et al. (1996) Proc.
31. TNT
Coupled Reticulocyte
Lysate Systems Technical
Bulletin #TB126, Promega
Corporation.
diseases, such as hereditary breast cancer (24),
7095% of the mutations that cause disease
result in a truncated gene product.
The PTT involves rst purifying genomic DNA or
mRNA from the patients blood or tissue. This is
followed by either RT-PCR
(f)
or PCR
(f)
with the
concurrent incorporation of a T7 promoter and
optimal translation initiation sequence
surrounding the desired start codon (25). Often
when the source of mRNA is limiting, a second
nested PCR amplication is required. Large
exons are amplied from genomic DNA while
smaller exons are amplied together from mRNA
and the gene is segmented into overlapping
amplied fragments. The amplied DNA is
added directly to a coupled transcription/trans-
lation reaction and translation terminating
mutations are detected as faster migrating
bands after SDS-PAGE analysis. PTT has the
advantage of enabling scans of large (23kb)
DNA/RNA segments quickly. In addition, PTT
detects only disease-causing mutations. This
avoids the fruitless evaluation of polymorphisms.
The recent introduction of the TNT
T7 Quick for
PCR DNA System
(c,d,e,g)
facilitates PTT analysis.
High-Throughput Screening(26)
Screening for Viral-Specic Translation Inhibitory
Compounds. Viruses contain a number of
different genetic elements used for promoting
viral expression at the expense of host mRNA
translation. Several groups are currently devel-
oping screens using in vitro expression of gene
constructs containing a viral element such as
the 5-UTR that can harbor an IRES (Internal
Ribosome Entry Site) followed by a rey or
Renilla luciferase gene (27). Chemical or
antibiotic libraries can be screened for specic
translation-inhibiting effects. The viral element
can be placed between the rey and Renilla
luciferase genes with translation of the rst gene
relying on normal cap-dependent initiation. Use
of the two luciferase genes allows normalization
of the second reporter behind the viral element.
The efcacy of compounds can be assessed
rapidly (<30 seconds) by assaying light output
from both the reporter and the control luciferase
(Figure 1, Chapter 5).
As a variation on this theme, RiboGene, Inc., has
reported developing a high-throughput system
for screening several hundred thousand
compounds for the ability to diminish or block
the required ribosomal frameshifting used
during translation of the HIV gag-pol mRNA.
This screen utilizes a reporter gene in which
luciferase (and light) is produced only when the
frameshift occurs.
Screening for Chaperonin-Inhibiting Drugs. The
in vitro luciferase folding/chaperonin assay
described earlier has been extended to under-
standing the role of heat shock factors, such as
Hsp90. It is now understood that disruption of
the folding pathways can result in proteolytic
degradation. Several groups are currently using
this information to ascertain the pharmacological
activities of benzoquinone ansamycins, such as
geldanamycin (28,29). These potentially
medically important compounds were rst
identied as interesting because of their ability
to inhibit tyrosine kinase activity. This ability
appears to be due to their interaction with
Hsp90, which prevents the correct folding of
tyrosine kinases and is followed by their prote-
olytic degradation. Other potentially important
drugs affecting protein folding through inhibition
of chaperonin function could be identied using
this approach.
Identication of Novel Orphan Receptors. The
binding of ligands to in vitro synthesized
receptors can be an important aspect of identi-
fying new receptors. For example, in a search
for novel orphan nuclear receptors and
ligands, a novel estrogen receptor was cloned
and characterized (30). Saturation ligand-
binding and ligand-competition assays of the in
vitro expressed clone allowed this novel
receptor to be distinguished from a previously
cloned receptor.
Functional Genomics
In Vitro Expression Cloning (IVEC). In this
procedure, an oligo(dT)-primed cDNA library is
constructed in a high copy expression plasmid
containing a T3, T7 or SP6 promoter. The
plasmid library is then transformed into E. coli,
and approximately 10
5
independent transfor-
mants are plated on selective media. The
bacterial colonies are grown to a specic size
(e.g., 1mm in diameter), collected and pooled
(50100 clones per pool). Puried plasmid DNA
from these pools is directly added to a small-
scale (e.g., 10l) coupled transcription/
translation reaction, where it is used as a
template in the presence of [
35
S]methionine (31).
Depending upon the number of the full-length
cDNA clones in the library, approximately 3050
proteins can be produced in a single reaction.
Proteins can be assayed for any number of
activities, including phosphorylation, proteolysis
or cleavage. Positive pools are subdivided until
the single cDNA that encodes the protein of
interest is isolated.
Ribosomal Display for Cell-Free Protein Evolution. In
this procedure, a cell-free system is used to
transcribe a DNA library, translate the mRNA
pools and, using a variety of techniques, the
proteins and the encoding mRNAs are retained,
still attached to the ribosomes. The protein-
mRNA-ribosome complexes are screened for
binding to a target, and the retained mRNA is
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32. Mattheakis, L.C., Bhatt, R.R.,
and Dower, W.J. (1994) Proc.
33. Hanes, J. and Plckthun, A.
(1997) Proc. Natl. Acad. Sci.
USA 94, 4937.
34. He, B., Gross, M., and
Roizman, B. (1996) Proc. Natl.
Acad. Sci. USA 94, 843.
35. Joyce, G.F. (1993) Pure Appl.
Chem. 65, 1205.
Preparative Synthesis
Large-Scale Protein Expression and Purication.
Cell-free expression systems are often preferred
over in vivo or native systems, because they can
be used for the expression of toxic, proteolyti-
cally sensitive or unstable proteins. In addition,
in vitro systems provide the ability to incorporate
non-natural amino acids containing photoacti-
vatable, uorescent or biotin residues. Typically,
in vitro systems produce nanogram amounts of
proteins per 50l reaction; however, preparative
scale methods have been developed recently
that may yield milligram quantities per milliliter of
reaction mixture.
Guide Organization
This chapter provides a general overview of
many of the major applications of in vitro
expression systems. The remainder of the guide
focuses on several of these applications,
seeking to provide more detailed information on
commonly used methodologies for in vitro
expression technology.
amplied using RT-PCR
(f)
with the resulting DNA
used for another round of selection. Initially, a
prokaryotic E. coli coupled transcription/trans-
lation system was used to generate large
libraries of peptides for receptor ligand
screening (32). Later improvements allowed
folding of whole proteins into their native
structure while still attached to the ribosome
(33). The rst eukaryotic application used a
coupled rabbit reticulocyte system to study
antibody-ribosome-mRNA (ARM) complexes,
allowing for rapid selection and monitoring of
antibody combining site evolution (34).
A cell-free system has been developed for
performing evolution studies in which RNA
amplication and the coupled reaction can be
performed simultaneously at a given temper-
ature (35). After unsuccessful attempts using
wheat germ extracts and coupled E. coli
systems, investigators were able to combine the
reactions using a rabbit reticulocyte coupled
system. By exerting selective pressure on
functional protein products necessary for RNA
amplication, this system can be used for
performing laboratory evolution.
P R O T E I N - P R O T E I N
I N T E R A C T I O N S
2
C H A P T E R
This illustration of protein-
protein interactions shows the
complementary t of two
protein molecules, such as a
ligand and its receptor
molecule on the cells surface.
7
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Chapter Two: Protein-Protein Interactions
Contents Page
Introduction .............................................................................................................. 8
Fusion Tag Approach .................................................................................................. 9
Immunoprecipitation.................................................................................................. 10
Far Western Analysis ................................................................................................ 11
Isolation of Protein Complexes by Capture
of Biotinylated Lysine Residues .................................................................................. 11
Protein Folding, Chaperonins and Luciferase .................................................................... 12
Real-Time Translation/Folding Assays ............................................................................ 12
Macromolecular Assembly and Frameshifting.................................................................... 12
Introduction
A popular current application of the coupled eukaryotic systems is detection of protein-protein inter-
actions. Often researchers will use the in vitro approach to verify or conrm in vivo results, such as
those obtained using the yeast two-hybrid approach (1). To dene the region of protein-protein inter-
action, usually a series of deletion constructs or occasionally specic point mutants are synthesized
in vitro and compared to wildtype, full-length proteins.
References
1. Boyd, J. et al. (1995)
Oncogene 11, 1921.
9
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C H A P T E R T W O
2. Chinnaiyan, A.M. et al. (1995)
Cell 81, 505.
3. Cowell, I. and Hurst, H. (1996)
4. Sharp, T.V., Witzel, J.E. and
Jagus, R. (1997) Eur. J.
Biochem. 250, 85.
5. Jagus, R. and Beckler, G.S.
(1998). Overview of eukaryotic
in vitro translation and
expression systems. Current
Protocols in Cell Biology
11.1.111.1.13. Copyright
1998 by John Wiley & Sons,
Inc. Reproduced by permission
of Wiley-Liss, Inc., a subsidiary
of John Wiley & Sons, Inc.
6. Pei, L. (1999) J. Biol. Chem.
274, 3151.
7. Chen, W. and Pei, L. (2000) J.
Biol. Chem. 275, 19422.
8. Cleveland, D.L. and Ihle, J.H.
(1995) Cell 81, 479.
.
Fusion Tag Approach
Gene products expressed in vivo as fusion or
tagged proteins can be used to detect pro-
tein-protein interactions or as an alternative to
antibody detection. Radioactive proteins can be
used as probes to detect interactions with sus-
pected protein partners that have been
expressed in E. coli as GST- (glutathione-S-
transferase), His- or epitope-tagged fusion
proteins (2). [
35
S]methionine-labeled proteins
can be synthesized using coupled in vitro reac-
tions from either full-length cDNAs or deletion
mutants. The fusion proteins can be bound to
an afnity matrix along with the radioactive pro-
teins with which they interact (3,4,5). The bound
radioactive proteins are then eluted and ana-
lyzed by SDS-PAGE or Western analysis
(Figure 1; 5).
Alternatively, a nonradioactive approach may be
used; the protein is labeled with biotinylated
lysine (e.g., Transcend Biotinylated tRNA) and
combined with a GST-tagged protein. The
biotinylated protein is detected by means com-
mon to Western blotting (6,7).
This fusion tag approach has been used to
study receptor-mediated control of apoptosis.
Binding of Fas ligand (FasL) or anti-Fas anti-
body to Fas (APO-1/CD95) receptor, or binding
of tumor necrosis factor (TNF) to the TNF recep-
tor (TNFR-1) rapidly induces cell death by an as
yet undetermined mechanism. A unique cyto-
plasmic motif present in both TNFR-1 and Fas,
the death domain, is necessary for induction
of cell death. The death domain is the site of
protein-protein interaction. The primary function
of FasL and TNF, as recently postulated, is to
mediate receptor aggregation (8). Therefore, a
critical step is to identify proteins that bind
directly to the cytoplasmic death domains of
these receptors.
FADD is a Fas-associated protein containing a
novel death domain that was identied by
Chinnaiyan et al. (2). Using Promegas TNT
T7
Coupled Reticulocyte Lysate System
(a,b,c,e)
(Cat.# L4610), these researchers synthesized
35
S-labeled FADD in vitro from a modied
expression vector. Labeled FADD was incubat-
ed with wildtype and mutant GST-Fas fusion
proteins as well as a GST-TNF fusion protein.
Figure 2A shows the ve GST fusion proteins
used in the binding assays. Figure 2B presents
the results of the binding experiment, illustrating
that FADD binds only the wildtype Fas construct
and the mutant Fas-FD5 construct. The Fas-FD5
construct encodes a protein with enhanced
apoptotic activity compared to that encoded by
the wildtype Fas.
Figure 1. The study of protein-protein interactions using the TNT
Systems (5). This schematic shows translation of one

protein with radioactive [
35
S]methionine in a TNT
System reaction. Large amounts of the suspected partner

protein are expressed and puried from E. coli. A fusion tag (most commonly GST) is incorporated into this
second protein to facilitate purication and subsequent capture steps. After the GST fusion protein is immobi-
lized on gluthathione-agarose, it is mixed with the protein produced in the TNT
reaction. The agarose is

washed to remove unbound protein and the remaining bound proteins are eluted and analyzed on a gel. This
technique allows quantitative measurement of the protein-protein interactions for both wildtype and mutant
proteins and is often used to verify the in vivo results obtained from yeast two-hybrid experiments.
35
S
Gene 1
TNT
System
Protein 1
GST Gene 1
Purify on Affinity Column Express in E. coli
GST Protein 2
Western
W-Wash
E-Eluate
M-Marker
Autoradiography
W E M W E M
2
5
9
3
M
A
0
3
_
9
A
10
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9. Benedict, C.M. and Clawson,
G.A. (1996) Biochemisty 35,
11612.
10. Leng, P., Brown, D.R. and Deb,
S. (1995) Int. J. Oncol. 6, 251.
11. Sif, S. and Gilmore, T.D. (1993)
J. Virol. 67, 7612.
12. Rice, N.R., MacKichan, M.L.
and Israel, A. (1992) Cell 71,
243.
13. Mercurio, F. et al. (1993) Genes
Dev. 7, 705.
formed spleen cells can be reconstituted in
vitro (5,11).
Proteins in the NF-B transcriptional activator
family act as tertiary messengers, transducing
signals from the environment to the nucleus of
the cell. NF-B resides in the cytoplasm as an
inactive complex consisting of heterodimeric
DNA-binding subunits sequestered by an
inhibitor (I-B). When an appropriate signal is
received, I-B is thought to be phosphorylated
and then dissociates from the DNA-binding
subunits, allowing the subunits to translocate
to the nucleus and bind their target genes.
In one pathway, the NF-B precursor proteins
p105 and p98 can form heterocomplexes with
both the NF-B subunit p65 (RelA) and the
proto-oncogene product c-Rel. This results in
the retention of these heterocomplexes in the
cytoplasm (12,13). When p105 or p98 is prote-
olytically processed to yield p50 or p55, the
DNA binding subunits are released from the
heterocomplexes and are free to translocate to
the nucleus or to interact with I-B.
Immunoprecipitation (5)
This approach utilizes antibodies against
a particular antigenic domain to detect a
radioactive fusion partner. For example, an
inuenza hemagglutinin (HA) epitope incorpo-
rated in the carboxyl terminus of an in vitro
expressed protein can be immunoprecipitated
using anti-HA antibodies (9). Alternatively, if an
antibody against one of the partners is avail-
able, then co-immunoprecipitation can be
used for detection (4,10). Again, the bound
radioactive proteins are then eluted and ana-
lyzed by SDS-PAGE or Western blot analysis.
A variation of this type of analysis uses in vitro
expression of several proteins simultaneously
in a coupled system. The relative protein
expression levels can be controlled by varying
the amount of each DNA construct. For
instance, experiments using cDNAs for chick-
en NF-B p105, NF-B p100, c-Rel, and v-Rel,
cotranslated in vitro, followed by protein com-
plex detection by immunoprecipitation with
specic antiserum, show that one of the
demonstrated complexes from v-Rel-trans-
GST
KRKEV
hFas +
319
V
238
Cytoplasmic Domain
Fusion FADD
protein binding
KDITS
304
hFas-FD5 +++
EKIQT
296
hFas-FD8
N
238
319
hFas-LPR
RYQRW
426
hTNFR-1 1
4
5
7
M
A
0
4
_
6
A
FADD
GST
fusions
G
S
T
G
S
T
-
T
N
F
R
-
1
G
S
T
-
F
A
S
-
F
D
8
G
S
T
-
F
A
S
-
L
P
R
G
S
T
-
F
A
S
-
F
D
5
G
S
T
-
F
A
S
Figure 2. Specic interaction of GST-Fas and GST-Fas-FD5 with in vitro translated FADD and FADD expressed in trans-
fected 293T cells. Panel A: Schematic representation of the GST fusion proteins containing the cytoplasmic domains
of Fas, Fas mutants and TNFR-1. Amino acid residues are given for selected junctions, and numbering is based on
the mature form of the receptor. The lymphoproliferation (lpr) mutant of Fas is represented (V
238
N
238
). Gray shad-
ing represents the death domain of Fas. Binding of FADD to the various GST fusion proteins is depicted at the right
and is based on data from Panel B. Panel B: Interaction of in vitro translated,
35
S-labeled FADD with various GST
fusion proteins immobilized on glutathione-Sepharose
beads. After the beads were washed, retained FADD

protein was analyzed by SDS-PAGE and autoradiography (upper panel). The gel was Coomassie
-stained,
and the bands representing the various GST fusion proteins were aligned to show equivalency of loading.
1
4
4
9
G
A
0
4
_
6
A
A.
B.
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C H A P T E R T W O
14. DiDonato, J.A. and Karin, M.
(1993) Promega Notes 42,16.
15. Johnston, S., Yu, X.M. and
Mertz, J.E. (1996) J. Virol.
70,1191.
16. Beckler, G. and Hurst, R.
17. Sanford, J.C. et al. (1995) J.
Biol. Chem. 270, 26904.
18. Rothschild, K.J., Sonar, S.M.
and Olejnik, J. (1997) U.S.
Pat. No. 5,643,722.
DiDonato and Karin (14) used immunoprecipi-
tation to demonstrate that the p98 and c-Rel
proteins are capable of interacting in vitro. In
the experiment shown in Figure 3, a c-Rel-spe-
cic antibody was used to immunoprecipitate
c-Rel from TNT
reactions expressing either

p98, c-Rel or both of these proteins. p98 was
coprecipitated when expressed in the pres-
ence of c-Rel (lanes 13) but not when
expressed alone (lane 5).
Far Western Analysis (5)
A direct detection method for identifying protein-
protein interactions after transfer of proteins
from polyacrylamide gels to a membrane has
been termed Far Western analysis. In this
approach, radioactive proteins are synthesized
in vitro and then used as probes to directly
detect binding to membrane-bound, renatured
proteins (15).
Isolation of Protein Complexes by Capture
of Biotinylated Lysine Residues (5)
One drawback of fusion protein techniques is
the requirement to make the fusion protein con-
struct. Another approach that obviates the need
for the development of novel constructs is to
incorporate non-natural amino acids, such as
biotinylated lysine residues into the in vitro
translated proteins. This approach will work for
any gene containing lysine codons. The incor-
poration of non-natural amino acids does not
signicantly alter the efciency of polypeptide
synthesis, and often incorporation of biotinylat-
ed lysines does not affect the function of the
protein (16). This approach has been used to
develop a novel method for capturing protein
complexes that associate with biotinylated
Rab5, a member of the Rab family of GTP-bind-
ing proteins (17). The ability to bind biotinylated
lysine tightly to streptavidin-linked agarose can
be utilized to capture in vitro synthesized
biotinylated Rab5. A promising and potentially
powerful modication of the biotinylated-lysine
capture technique proposes using photocleav-
able (PC)-biotin for the detection and gentle
purication of in vitro generated polypeptides
(18). For example, the capture of the PC-biotin
nascent polypeptides using streptavidin-coated
magnetic beads has been described. After a
short exposure to UV light, the nascent
p98
1 2 3 4 5
Antiserum
anti-c-Rel + + + + +
Template DNA
p98 ++++ ++ + +
c-rel + + + +
c-Rel
Figure 3. Protein-protein binding studies using proteins co-expressed in the TNT
Wheat Germ System.

35
S-labeled
c-Rel and p98 were produced in 25l TNT
Wheat Germ Extract

(a,b,c,e)
reactions containing 1.5g of c-rel
template DNA (lanes 14) and 1.0g, 0.5g, 0.25g or 0.25g of p98 template DNA (lanes 1, 2, 3 and 5,
respectively). A 4l aliquot from each reaction was analyzed by immunoprecipitation with anti-c-Rel antiserum
as described in reference 10 and then analyzed by 10% SDS-PAGE. For the gel shown in this gure, lanes
13 were loaded to contain equivalent amounts of p98. The immunoprecipitation results were detected by
autoradiography after 16 hours at 70C.
0
0
8
6
T
A
0
4
/
3
A
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19. Olejnik, J. et al. (1995) Proc.
20. Olejnik, J., Krzymanska-
Olejnik, E. and Rothschild, K.J.
(1998) Meth. Enzymol. 291,
135.
21. Bazzoni, G. et al. (2000) J. Biol.
Chem. 275, 20520.
22. Frydman, J. et al. (1994)
Nature 370, 111.
23. Frydman, J. and Hartl, F.U.
(1996) Science 272, 1497.
24. Kolb, V.A., Makeyev, E.V. and
Spirin, A.S. (1994) EMBO J. 13,
3631.
25. Makeyev, E.V., Kolb, V.A. and
Spirin, A.S. (1996) FEBS Lett.
378, 166.
26. Sakalian, M. et al. (1996) J.
Virol. 70, 3706.
the ribosome (24). However, no luciferase activi-
ty was observed, while full-length luciferase
remained attached to the ribosome as a pep-
tidyl-tRNA, probably because the C-terminal
portion of the enzyme is masked by the ribo-
some and/or ribosome-associated proteins. The
investigators demonstrated that the ribosome-
bound enzyme acquires enzymatic activity
when its C-terminus is extended by at least 26
additional amino acid residues (25). The results
demonstrate that the acquisition of the nal
native conformation by a nascent protein can
occur as the protein is being synthesized and
that folding does not require release of the pro-
tein from the ribosome.
Macromolecular Assembly and
Frameshifting (5)
Many in vivo translational control mechanisms
are faithfully replicated in vitro. In addition, a
variety of macromolecular complexes can be
expressed and properly assembled in vitro. For
example, an in vitro synthesis and assembly
system for the protypical type D retrovirus,
Mason-Pzer monkey virus (M-PMV), has been
developed. This system uses rabbit reticulocyte
reactions expressing M-PMV Gag precursor
polyproteins as the result of two ribosomal
frameshift events (26). The frameshift efciency
in vitro is identical to that observed in vivo.
These polyproteins assemble to form immature
retrovirus capsids indistinguishable from those
formed in the host cell cytoplasm. More impor-
tantly, this system can be utilized for the analysis
for potential inhibitors of retrovirus assembly with
the use of anti-Gag antibodies.
polypeptide was released (7095% efciency)
in native form with no remaining tags (1820).
In another procedure, the ZO-1 protein and its
role as a tight junction component was studied
by using the TNT
Coupled Reticulocyte Lysate

System
(a,b,c,e)
and Transcend
Biotinylated
tRNA (21).
Protein Folding, Chaperonins and
Luciferase (5)
In vitro expression is increasingly being used to
understand the nature of sequential chaperonin
interactions required for protein folding.
Researchers in this eld have combined the
advantages of in vitro expression with the power
of instantaneous reporter gene product assays.
The folding of polypeptides emerging from ribo-
somes has been analyzed using rey
luciferase as a model protein (22,23). The grow-
ing polypeptide interacts with a specic set of
molecular chaperones, including Hsp70, the
DnaJ homologue Hsp40 and the chaperonin
TRiC. The ordered assembly of these compo-
nents on the nascent chain forms a high
molecular mass complex that allows the
cotranslational formation of protein domains and
the completion of folding once the chain is
released from the ribosome.
Real-Time Translation/Folding Assays (5)
A novel approach has been developed using a
wheat germ system in which the components
for the luciferase enzymatic assay have been
added directly to the translation reaction and
monitored continuously in real time (24). In
order to demonstrate that luciferase exhibits
cotranslational folding, a comparison was made
of the activity of translation products produced
from wildtype mRNAs with those produced from
mutant mRNAs lacking stop codons to prevent
release of the polypeptide from the ribosome.
Luciferase was shown to be completely folded
and fully active immediately upon release from
P R O T E I N N U C L E I C
A C I D I N T E R A C T I O N S
3
C H A P T E R
In this illustration protein-DNA
binding is shown. One
example of such an interaction
is the binding of transcription
factors to DNA, which results
in regulation of DNA synthesis.
13
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Chapter Three: Protein-Nucleic Acid Interactions
Contents Page
Introduction ............................................................................................................ 14
Protein-DNA Interactions ............................................................................................ 15
Protein-RNA Interactions ............................................................................................ 15
Introduction
Cell-free systems have become popular tools for in vitro production of proteins, examined for their
activity in protein-nucleic acid interactions. Putative DNA binding proteins, such as transcription
factors, can be analyzed for their ability to bind specic sequences on radiolabeled DNA or RNA.
Generally binding of proteins to nucleic acids is detected by electrophoretic mobility shift assay
(EMSA). Using this assay, protein-nucleic acid complexes demonstrate retarded migration compared
to their non-nucleic acid-binding counterparts.
15
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C H A P T E R T H R E E
Protein-DNA Interactions
One of the most commonly used procedures for
detecting DNA-binding proteins or protein
complexes (such as transcription factors) is the
electrophoretic mobility shift assay (EMSA) or
gel shift assay. This popular technique uses in
vitro expression to synthesize putative DNA
binding proteins, which are then incubated with
an oligonucleotide containing a target
consensus sequence site. Binding to the DNA is
detected by gel shift assay (Figures 1 and 2).
Either partner can be radiolabeled (usually 5-
end-labeled [
32
P]DNA), and with proper
controls, the translation extract containing the
synthesized candidate binding factor can be
used directly in the assay (1).
When c-Rel was cotranslated with a small
amount of p98, very little inhibition of c-Rel
binding to DNA was observed (Figure 2, lane 3).
However, when a 4-fold higher ratio of p98:c-Rel
DNA was used in the TNT
System co-
expression, and the binding reaction was then
performed using an amount of c-Rel equivalent
to that in lane 3, no c-Rel-DNA complex was
observed (Figure 2, lane 4). Thus, p98 acts as
References
1. Lee, H.J. and Chang, C. (1995)
J. Biol. Chem. 270, 5434.
2. Mercurio, F. et al. (1993) Genes
Dev. 7, 705.
3. Sif, S. and Gilmore, T.D. (1993)
J. Virol. 67, 7612.
4. Ebel, T. and Sippel, A. (1995)
5. Di Donato, J. A. and Karin, M.
.
an I-B in vitro by preventing c-Rel from binding
DNA. This in vitro interaction is similar to the
ability of p98 to retain c-Rel in the cytoplasm in
vivo (2).
Many researchers investigating transcription
factor binding use the coupled wheat germ
translation systems rather than rabbit reticu-
locyte systems, as the wheat germ extract
does not contain endogenous mammalian
transcription factors, such as NF-B (3). If the
reticulocyte system is used, a rapid method to
remove endogenous DNA-binding proteins from
the reticulocyte system has been developed
using biotinylated DNA and streptavidin
magnetic beads (4).
Protein-RNA Interactions
Many of the techniques using in vitro translated
proteins to detect DNA-binding proteins can be
used to study protein-RNA interactions as well.
However, one of the ways in which the
techniques differ is in the ability to produce a
number of modied RNAs using in vitro
transcription. In one example, the direct inter-
action of the U1 snRNP-A protein with SV40 late
Gene 1
Protein 1
oligo
Autoradiography
32
P
o
l
i
g
o
o
l
i
g
o
/
P
r
o
t
e
i
n
w
i
t
h

c
o
l
d

o
l
i
g
o
32
P
32
P
32
P
32
P
Protein-DNA
Complexes
TNT System
2
6
0
2
M
A
0
3
/
9
A
Figure 1. Use of the TNT
Systems for studying protein-DNA and protein-RNA interactions. An unlabeled (non-

radioactive) protein is produced in a TNT
System reaction and then mixed with a

32
P-labeled
oligonucleotide containing a consensus sequence for the suspected DNA binding protein (e.g., a
transcription factor target sequence). The protein-DNA complexes are analyzed by migration on
polyacrylamide gels. Unbound oligonucleotide will migrate to a position near the bottom of the gel. The
oligonucleotide-protein complex will show retarded mobility and will migrate to a position near the top
of the gel. The mobility shift can be measured quantitatively. DiDonato and Karin demonstrated that
p98 sequesters c-Rel in the cytoplasm, thereby acting as an I-B (2). In the same experiment as
described in Chapter 2 (5), the functional signicance of this interaction in vitro was examined by gel
shift assays to determine the effect of p98 binding on the DNA-binding capacity of c-Rel. Figure 2
demonstrates that gel shift assays can be performed using single or multiple proteins expressed in a
TNT
reaction and added directly to the assay.

16
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6. Lutz, C.S. and Alwine, J.C.
(1994) Genes Dev. 8, 576.
7. Denman, R.B. (1998) Promega
Notes 67, 5.
8. Dubnau, J. and Struhl, G.
(1996) Nature 379, 694.
9. Zaidi, S.H., Denman, R. and
Malter, J.S.
(1994) J. Biol. Chem. 269,
24000.
10. Sen, R. and Baltimore, D.
(1986) Cell 46, 705.
11. Haskill, D. et al. (1991) Cell 65,
1281.
Next, the biotinylated binding protein is coupled
to a SoftLink
Soft Release Avidin

(h)
solid
support (8) making an afnity column. Total RNA
or poly(A)+ RNA (9) is then incubated with the
afnity resin in TBS buffer, allowing the protein/
target RNA interaction to occur. Unbound RNA
is removed by centrifugation, and after extensive
washing with TBS buffer, the bound RNA is
eluted from the resin under strong denaturing
conditions. This RNA, which contains the entire
subpopulation of RNA specically bound to the
biotinylated protein, can then be fractionated
and analyzed using differential display RT-PCR
(f)
(DDRT-PCR
(f)
), subtractive hybridization or
reverse Northern blotting (Figure 2).
Many of the in vitro protein modications used
for detection of protein-protein interactions can
be used for investigation of purported nucleic
acid-binding proteins. When combined with the
variety of standard DNA and RNA analysis
procedures, the options for identication and
characterization of these interactions increase
dramatically.
mRNAs was demonstrated by mixing [
35
S]Met
U1 snRNP-A fusion protein containing a g10
epitope tag synthesized in vitro and
32
P-labeled
viral RNA transcribed in vitro. The complexes
were then immunoprecipitated, and coprecipi-
tating RNAs were extracted and analyzed by
electrophoresis (6).
Denman (7) described another technique that
can be applied to binding studies of both DNA
and RNA. This technique, outlined in Figure 3,
uses a three-step scheme for isolating targets of
DNA- or RNA-binding proteins. In this example,
RNA was used. First, a biotinylated binding
protein or protein binding domain is synthesized
by incorporating biotinylated lysines (e.g.,
Transcend
Biotinylated tRNA, Cat.# L5061) in a

coupled transcription/translation reaction using
the TNT
System programmed with plasmid

DNA. At least one lysine residue is required for
biotinylated protein synthesis. In the unlikely
event of no naturally occurring lysine residues, a
lysine tag can be engineered at the end of the
protein or peptide fragment.
Template DNA
MAD-3 +
p98 + ++++
c-rel + + + + +
Probe Ig Ig Ig Ig Ig
Competitor Ig
c-RelDNA
complex
Ig probe
1 2 3 4 5
Figure 2. Gel shift assays using proteins produced in
the TNT
Wheat Germ System. c-Rel, p98 and

MAD-3 proteins were produced in unlabeled 25l
TNT
Wheat Germ Extract

(a,b,c,e)
reactions
containing 1.0g of c-rel template DNA (lanes
15), 0.25g or 1.0g of p98 template DNA
(lanes 3 and 4, respectively), and 1.0g of MAD-
3 template DNA (lane 5). The relative expression
levels of each protein were estimated using a
parallel set of TNT
reactions containing
[
35
S]methionine. Gel shift assays were performed
as described in reference 10. Based on the data
from the
35
S-labeled reactions, the volume of
unlabeled TNT
reaction products added to each

gel shift reaction was adjusted to contain a
constant amount of c-Rel (from 28l). Each
reaction also contained 40pg of
32
P-labeled Ig
enhancer probe DNA (10). In lane 2, the
unlabeled competitor Ig probe was added in 50-
fold excess (2ng). The gel shift reactions were
analyzed by electrophoresis on a 5% polyacry-
lamide gel and detected by autoradiography for
24 hours at 70C. c-Rel bound specically to
the immunoglobulin kappa light chain enhancer
probe (lanes 1 and 2) (10). As expected, this
binding could be inhibited by adding in vitro-
translated MAD-3 (I-B-, a 37kDa member of
the I-B family) (11) to the c-rel binding reaction
(lane 5).
0
0
8
7
T
A
0
4
_
3
A
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C H A P T E R T H R E E
12. TriReagent
RNA/DNA/Protein
Isolation Reagent Technical
Manual, Molecular Research
Center, Inc.
13. Sung, Y.-J. and Denman, R.B.
(1997) BioTechniques 23, 462.
14. pGEM
-T and pGEM
-T Easy
Vector Systems Technical
Manual #TM042, Promega
Corporation.
A A A
A A
P*
A
A A A
b-nonbinding
protein
b-binding
protein
1. 2. 3.
Total RNA RNA-bound RNA-bound RNA-bound
negative
control
negative
control
Add protein, allow to
bind to SoftLink
Soft
Release Avidin solid support
by incubating 1 hour at 4 C.
Incorporation of biotin into
protein during TNT Coupled
Transcription/Translation
System reaction.
Wash with TBS buffer,
30X column volumes.
Add 15g RNA (or DNA)
and incubate for 1 hour
at 4 C.
Remove unbound RNA and
precipitate or remove unbound
RNA in Tri-Reagent and
precipitate.
Amplify and label RNA by
DDRT-PCR using [-
32
P]dCTP.
b
b
b
no template
P
P* P
Figure 3. Isolating targets of RNA- and DNA-binding proteins. Biotinylated RNA- or DNA-binding proteins were
produced in a 25l TNT
Coupled Transcription/Translation System

(a,b,c,e)
reaction in the presence of 2l
Transcend
tRNA (12) and were bound to 100l of SoftLink
Soft Release Avidin Resin

(h)
(Cat.# V2011, V2012)
at 4C. Fifteen micrograms of total RNA or genomic DNA were added and allowed to bind for 1 hour. Unbound
nucleic acid was removed with 30 column washes using TBS buffer. Bound nucleic acid was then eluted in
TriReagent
(Molecular Research Center; 12) and puried by ethanol precipitation. One-third of the puried
products were converted to complementary ssDNA using one of the 3-DDRT-PCR
(f)
primers. A fraction (1/25) of
the complementary ssDNA sample was then amplied by DDRT-PCR in the presence of 2Ci [-
32
P]dCTP (13).
The products were sized on denaturing polyacrylamide gels and select bands were cloned (14) and
sequenced. To demonstrate specicity, two control reactions were run in parallel with the protein of interest, an
unrelated control protein (column 2) and transcription/translation reaction without plasmid DNA (column 3).
2
2
4
3
M
A
0
5
_
8
A
T H E P R O T E I N
T R U N C A T I O N T E S T
4
C H A P T E R
This diagram depicts
production of a truncated
protein (right) as compared
to a normal length protein
(left) using the Protein
Truncation Test. This test
has been used, in vitro, to
determine whether a gene
mutation results in a
shortened translation
product that may lead to a
cancerous cell.
18
19
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T H E P R O T E I N
C H A P T E R F O U R
Chapter Four: The Protein Truncation Test
Contents Page
Introduction ............................................................................................................ 19
PTT Principle .......................................................................................................... 20
Source Considerations................................................................................................ 21
Detection and Primer Design ........................................................................................ 22
References
1. Lesieur, A. et al. (1994) Diag.
Mol. Pathol. 3, 75.
2. Lee, K-O. et al. (1996) J.
Biochem. Mol. Biol. 29, 241.
3. den Dunnen, J.T. et al. (1989)
Am. J. Hum. Genet. 45, 835.
4. Chamberlain, J.S. et al. (1988)
5. Loenig, M. et al. (1987) Cell 50,
509.
6. Orita, M. et al. (1989) Genomics
5, 874.
Introduction
Mutations in a gene can range from large deletions to single point mutations. Many of the large
deletions or translocations can be readily detected. For example, 95% of the cases of chronic myel-
ogenous leukemia contain the Philadelphia chromosome, which is a translocation of part of
chromosome 22 to chromosome 9. The abnormality can be detected by Southern blotting as aber-
rant or additional reactive bands when compared to normal samples (1). In this translocation, the
abl proto-oncogene is translocated into the bcr gene resulting in the expression of a bcr-abl fusion
protein. The chimeric transcript can be readily detected by RT-PCR
(f)
(2). Point mutations or small
deletions, however, are much more difcult to detect. In Duchenne muscular dystrophy (DMD), for
example, one third of the reported mutations in the gene DMD are not detectable as intragenic dele-
tions or duplications (35). Techniques such as single strand conrmation polymorphism (6) can
detect sequence differences but cannot distinguish between a polymorphism that may result in no
phenotype (e.g., conservative amino acid change) and a polymorphism with a denite effect on the
protein produced (e.g., premature termination of sequence).
A rapid solution to these problems can be achieved through a procedure known as PTT (protein
truncation test).
20
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7. Roest, P.A.M. et al. (1993) Hum.
8. Baklanov, M.M. et al. (1996)
9. TNT
Quick Coupled
Transcription/Translation
Systems Technical Manual
#TM045, Promega Corporation.
10. Kozak, M. (1986) Cell 44, 283.
11. pGEM
-T and pGEM
-T Easy
Vector Systems Technical
Corporation.
*For Laboratory Use.
PTT Principle
A simple way to judge whether a mutation
results in a truncation or not, is to translate the
protein in vitro. Roest et al. (7) developed the
protein truncation test (PTT) to rapidly screen for
these mutations. PTT is composed of four steps:
i) isolation of nucleic acid, either genomic DNA,
total RNA or poly (A)+ RNA; ii) amplication of a
specic region of the gene of interest; iii) in vitro
transcription and translation of the product of the
amplication reaction; and iv) detection of the
translation products. The shorter protein
products of the mutant alleles are easily distin-
guished from the full-length protein product of
the normal allele (Figure 1). PTT has been used
to analyze many genes in addition to DMD
(Table 1).
Amplied sequences for PTT can be generated
across the entire protein coding sequence or
they can be generated to specic exons. The
key feature of PTT is a specically designed
PCR primer to allow coupled in vitro transcrip-
tion/translation of the amplied sequence. The
primer contains a T7 bacteriophage promoter
sequence at the 5-end that directs transcrip-
tion. Usually, additional nucleotides are present
upstream of the T7 promoter. Even the addition
of a single G nucleotide upstream of the pro-
moter increases the transcriptional efciency
(8). While T7 is the most commonly used pro-
moter, T3 RNA polymerase promoter can be
used as well. SP6 promoters are not well-suited
for coupled transcription/translation of linear
DNA (9). Promega offers a system specically
for the expression of PCR
(f)
products, the TNT
T7 Quick for PCR DNA

(c,d,e)
(Cat.# L5540)*. A
36bp spacer separates the promoter
sequence from an optimal eukaryotic translation
initiation sequence, which includes the initiation
codon ATG. The optimal eukaryotic translation
initiation sequence is referred to as a Kozak
consensus sequence (10). The bacteriophage
promoter, spacer and Kozak sequence are fol-
lowed by sequences specic to the target
(Table 2). At the 3-end of the target, the primer
can include a stop codon if the amplied
sequence does not contain the native stop
codon (9). Restriction enzyme recognition sites
can also be engineered into both primers to aid
Figure 1. Schematic diagram of the Protein Truncation Test.
+ Forward Primer
RNA
Reverse Primer
ATG
T7
ATG
AUG
T7
Genomic DNA
Reverse
Transcription
PCR
Protein
Agarose gel electrophoresis
of PCR products
Full-length protein
Truncated protein
SDS-PAGE plus autoradiography
Cells from blood
or tissue sample
mRNA
cDNA
dsDNA
in vitro
Transcription/
Translation
PCR
dsDNA
1 2 3 4 5
Exons
1 2 3 4 5
Exons
RNA
1
7
7
0
M
A
0
4
_
7
B
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T H E P R O T E I N
C H A P T E R F O U R
12. Hogervorst, F.B.L. et al. (1995)
13. Lancaster, J.M. et al. (1996) Nat.
Genet. 13, 238.
14. Powell, S.M. et al. (1993) New
Eng. J. Med. 329, 1982.
15. van der Luijt, R. et al. (1994)
Genomics 20, 1.
16. Dang, M. et al. (1998) Lab.
Invest. 78, 195.
17. van Bakel, I. et al. (1997) Hum.
18. Hogervorst, F.B.L. (1997)
Promega Notes 62, 7.
19. TranscendNon-Radioactive
Translation Detection Systems
Technical Bulletin #TB182,
Promega Corporation.
20. Kirchgesser, M. et al. (1998)
Clin. Chem. Lab. Med. 36, 567.
21. Rowan, A.J. and Bodmer, W.F.
(1997) Hum. Mutat. 9, 172.
22. Eccles, D.M. et al. (1996) Am. J.
Hum. Genet. 59, 1193.
23. Wright, J. et al. (1996) Am. J.
Hum. Genet. 59, 839.
24. Bai, M. et al. (1997) J. Clin.
Invest. 99, 1917.
25. Romey, M. et al. (1996) Hum.
Genet. 98, 328.
26. Beaufrre, L. et al. (1997) Exp.
Eye Res. 65, 849.
27. Gardner, R.J. et al. (1995) Am.
J. Hum. Genet. 57, 311.
28. Lo Ten Foe, J.R. et al. (1996)
29. Pegoraro, E. et al. (1998)
Neurology 51, 101.
30. Liu, B. et al. (1994) Cancer Res.
54, 4590.
31. Papadopoulos, N. et al. (1994)
Science 263, 1625.
32. Heim, R.A. et al. (1994) Nat.
Genet. 8, 218.
33. MacCollin, M. et al. (1994) Am.
J. Hum. Genet. 55, 314.
34. Axton, R. et al. (1997) J. Med.
Genet. 34, 279.
35. Maugard, C. et al. (1997) Br. J.
Haematol. 98, 21.
36. Roelfsema, J.H. et al. (1996)
Nephrol. Dial. Transplant.
11(suppl. 6), 5.
vidual exons must be analyzed. To analyze the
entire coding sequence of a gene like BRCA1,
24 individual exons would need to be amplied
and analyzed. Besides requiring a large num-
ber of amplications, assuming all the exons are
large enough to translate, analysis of the indi-
vidual exons could miss truncation mutations
that could result in aberrant exon splicing. In the
same study that amplied exon 11 of the
BRCA1 gene from genomic DNA for PTT analy-
sis, Hogervorst et al. (12) isolated total RNA
from freshly isolated peripheral blood lympho-
cytes. The sequences corresponding to exons
210 were amplied by RT-PCR
(f)
and analyzed
by PTT. One subject had a mutation in one
allele that resulted, rst, in a smaller RT-PCR
product and, second, in a truncated protein by
PTT. The mutation was directly sequenced and
resulted from aberrant splicing of exons 9 and
10. Thus, using RT-PCR and PTT, larger portions
of a gene can be amplied and analyzed, pick-
ing up aberrant splicing mutations not identied
by analysis of the exons via amplication of
genomic DNA. In most cases, when RT-PCR is
used as the method to generate targets, the
entire coding region is broken into several
smaller fragments. For example, three amplica-
tions were used to test the entire coding region
of the TSC2 gene by PTT (17). When using mul-
tiple targets to span an entire coding region, the
amplimers should overlap so that a mutation at
the 3-end of one target (that does not cause a
in subcloning of the PCR product if verication
of a mutation is needed. The advent of PCR
product cloning vectors has abrogated the
need for inclusion of the restriction sites into
PCR primers (11).
Source Considerations
The PTT test can be applied to individual exons
of a gene via amplication of genomic DNA.
Hogervorst et al. (12) analyzed genomic DNA of
stored heparinized blood for mutations in the
breast and ovarian cancer gene, BRCA1.
Greater than 75% of the reported mutations in
BRCA1 result in truncated proteins. Primers
were designed to amplify exon 11, which
encodes 61% of the BRCA1 gene product.
Members of 35 families were analyzed, and all
produced the correct size of PCR
(f)
product from
the exon. The PCR product was transcribed
and translated in vitro with [
35
S]methionine and
analyzed by SDS-PAGE and autoradiography.
Six mutations resulting in truncated proteins
were identied. The mutant PCR products were
directly sequenced and were found to be the
result of either insertions or deletions yielding
frameshift mutations and premature stop
codons. Genomic DNA has also been used to
analyze the genes BRCA2 (13), APC (14,15)
and PLEC1 (16) by PTT.
Use of genomic DNA as the source of nucleic
acid for PTT has some drawbacks in that indi-
Table 1. Genes Analyzed with the Protein Truncation Test
a
.
Condition Gene Ref.
Familial Adenomatous Polyposis APC 14,15
Hereditary Desmoid Disease APC 22
Ataxia Telangiectasia ATM 23
Hereditary Breast and Ovarian Cancer BRCA1 12
BRCA2 13
Familial Hypocalciuric Hypercalcemia CASR 24
Cystic Fibrosis CFTR 25
Chorioderemia CHM 26
Duchenne Muscular Dystrophy DMD 7,27
Fanconi Anaemia FAA 28
Congenital Muscular Dystrophy laminin-2 29
Hereditary Non-Polyposis Colorectal Cancer hMSH2 30
hMLH1 31
Neurobromatosis Type 1 NF1 32
Neurobromatosis Type 2 NF2 33
Aniridia PAX6 34
Paroxysmal Nocturnal Haemoglobinuria PIG-A 35
Polycystic Kidney Disease PKD1 36,37
Epidermolysis Bullosa with Muscular Dystrophy PLEC1 16,4446
Dystrophic Epidermolysis Bullosa COL7A1 43
Breast Cancers, Gliomas, Melanomas PTEN/MMAC1 38,39,40
Rubenstein-Taybi Syndrome RTS 41
Familial Tuberous Sclerosis TSC2 17
a
More references available in The Protein Truncation Test Bibliography (BL002) and Mutation Detection (BR043) also available on the Internet at www.promega.com
22
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37. Roelfsema, J.H. et al. (1997)
Am. J. Hum. Genet. 61, 1044.
38. Li, J. et al. (1997) Science 275,
1943.
39. Furnari, F.B. et al. (1997) Proc.
40. Robertson, G.P. et al. (1998)
Proc. Natl. Acad. Sci. USA 95,
9418.
41. Petrij, F. et al. (1995) Nature 376,
348.
42. Sarkar, G. and Sommer, S.S.
(1989) Science 244, 331.
43. Whittock, N.V. et al. (1999) J.
Invest. Dermatol. 113, 673.
44. Takizawa, Y. et al. (1999) J.
Invest. Dermatol. 112, 109.
45. Kunz, M. et al. (2000) J. Invest.
Dermatol. 114, 376.
46. Rouan F. et al. (1989) J. Invest.
Dermatol. 114, 381.
signicant change in molecular weight) will be
detected in another target having the same
codons near the 5-end.
Detection and Primer Design
The detection method for PTT products must be
considered when designing primers for ampli-
cation (18). Typically, [
35
S]methionine is the
label of choice but other labels such as
[
35
S]cysteine and [
3
H]leucine could be used as
well. Thus, the amplied segments should con-
tain one or more of these amino acids. The
reactions are resolved on an SDS-PAGE gel and
either directly dried or uorographically
enhanced and exposed to X-ray lm (9). The
dried gels can also be analyzed by phospho-
rimaging. When radioactive incorporation is not
an option, non-radioactive techniques are avail-
able. Proteins can be tagged with biotin by
inclusion of biotinylated lysine tRNA in the trans-
lation reaction (9,19). The biotin moiety is then
detected with a streptavidin-enzyme conjugate
and developed via either a colorimetric or
chemiluminescent reaction (19). For example,
PTT has been applied to the APC gene using
translation with a biotinylated lysine tRNA (20).
Other methods for non-radioactive detection
include the inclusion of an epitope tag in the 5-
primer so that the translation products can be
analyzed by Western blotting with an antibody
that binds the epitope (21). When dealing with a
heterozygous condition, both the normal and
mutant targets will be amplied and both the
truncated and full-length protein will be detect-
ed, unless the allelle is on the X or Y
chromosome of male subjects, no matter which
detection method is chosen.
PTT offers a quick and easy method for analyz-
ing a protein coding sequence for truncation
mutations. However, the method has some limi-
tations. If the truncated sequence does not
translate well or does not contain the appropri-
ate amino acid for labeling, the mutation could
be overlooked. Also, if the truncation is very
near the 3-end of the target, truncation could
be missed due to the inability of SDS-PAGE to
resolve such differences. If the mutations are
very near the 5-end of the coding sequence,
the mutation could be missed as well.
Renements of PTT detection, such as the
incorporation of an epitope tag into the 5 PCR
primer (21), could allow detection of these
mutations, since incorporation of a specic
amino acid is not needed for detection. Finally,
incorporation of uorescence-tagged amino
acids may simplify the detection of proteins by
PTT and can possibly be used for quantitation
of the mutant protein (18).
Table 2. Sequences of Different T7-Modied Oligonucleotide Primers for In Vitro Transcription
and Translation
a
.
Eukaryotic
Translation
Restriction T7 Bacteriophage Initiation
Site Sequence Sequence Spacer Sequence Ref.
GGATCC TAATACGACTCACTATAGGG AG CCACC ATG 13,42
GGATCC TAATACGACTCACTATAGGG AG CCACC ATG G 30,31
GGATCC TAATACGACTCACTATAGG AACAG CCACC ATG 7,15
nnn
b
TAATACGACTCACTATAGG AACAG CCACC ATG G 12,28
a
Sequences provided are for only the upstream portion of the 5 primer that is not gene specic. For gene-specic use, the eukaryotic translation
initiation sequence would be followed by 1720 bases exactly complementary to the sequence of interest.
b
n = any nucleotide
S C R E E N I N G
A P P L I C A T I O N S
5
C H A P T E R
This illustration represents a
small organic molecule, for
instance one produced by
combinatorial chemistry
techniques, that is being
screened for its t to the
larger protein molecule. The
inhibition of function of the
protein molecule upon
attachment by the organic
molecule would signify the
identication of a successful
drug candidate or target.
23
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Chapter Five: Screening Applications
Contents Page
Introduction ............................................................................................................ 24
Monitoring and Detection ............................................................................................ 26
Model Enzyme Systems .............................................................................................. 26
Development of Antiviral Agents.................................................................................... 27
Antisense Regulation ................................................................................................ 27
Non-Isotopic Detection................................................................................................ 27
Ultra-Sensitive DNA Detection and Immunoassays.............................................................. 28
Expression Immunoassay ............................................................................................ 28
Introduction
Pioneering work leading to the elucidation of the genetic code and the characterization of the protein
synthesis machinery relied heavily upon in vitro transcription and translation systems. Such systems
allow the use of dened templates to direct the synthesis of proteins and permit the direct analysis of
the transcription and translation machinery. The emergence of combinatorial chemistry techniques
and the need for new antibiotics to overcome increasing bacterial resistance has brought the basic
tool of in vitro translation systems back intothe spotlight. Both prokaryotic and eukaryotic in vitro trans-
lation systems have found great utility in efforts to screen organic compounds for inhibition of the
basic cellular functions of transcription and translation, common targets for antibiotic compounds
(Figure 1).
In vitro transcription and translation systems can provide some advantages over in vivo systems for
screening purposes. In vitro systems allow exact manipulation of compound concentrations. This is
an important parameter when evaluating the potential potency of the lead compound. There is no
need for cellular uptake to evaluate the effect of the compounds. While uptake evaluation is important
for determining the eventual efcacy of the drug, it can unnecessarily eliminate valuable lead
compounds in an initial screen. The interpretation of results in living cells is complicated by the large
number of intertwined biochemical pathways and the ever-changing landscape of the growing cell. In
vitro systems allow the dissection of effects in a static system for simpler interpretation of results and
the ability to specically monitor individual processes such as transcription or translation. Individual
targets not normally present, or found at low concentrations, can be added in controlled amounts.
Beyond the basic transcription and translation machinery found in the extracts themselves, target
proteins for screening can be generated from genetic material by simple in vitro expression from
added DNA templates. In short, in vitro transcription and translation systems provide a exible and
consistent tool for screening.
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S C R E E N I N G
C H A P T E R F I V E
Figure 1. In vitro expression screening
of a small molecule library for
compounds that specically inhibit
virus-directed translation, using a
Dual-Luciferase
readout assay.
The diagram depicts transcription
and translation of the rey
luciferase gene (luc) containing nor-
mal cellular translation signals (5
untranslated region and Kozak initia-
tion sequence) followed by the
Renilla luciferase gene (Rluc) direct-
ed by a viral translation initiating
sequence (IRES, internal ribosome
entry site). Potential small molecule
inhibitors are tested for the ability to
specically inhibit the viral-based
translation of the Rluc, while not
affecting the normal cellular transla-
tion of luc. The Luciferase Assay
Reagent specically produces light
from the rey luc, followed by the
addition of Stop & Glo
Reagent,
which immediately turns off enzy-
matic light production from luc and
activates expression from Rluc.This
dual-reporter assay is very powerful
for detecting viral specic inhibitors
that do not affect normal cellular
translation.
Transcription
Translation
TNT
5
5
Luciferase
Assay Reagent
Stop & Glo
Reagent
luc
Rluc
Allows
Translation
Blocks
Translation
Rluc
Rluc
Viral
IRES
Viral
IRES
luc
luc
T7
Library
luc
Rluc
Specific viral translation
inhibitor from library.
No effect on cellular
or viral translation.
No light produced
D u a l - L u c i f e r a s e
A s s a y R e a g e n t
3
0
7
1
M
A
0
9
_
0
A
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References
1. Yike, I. et al. (1999) Appl.
Environ. Microbiol. 65, 88.
method of screening is using RRL and
luciferase to screen environmental samples for
trichothecene mycotoxins, potent translation
inhibitors (1). This reaction differs from transcrip-
tion, which uses a DNA template encoding the
reporter gene. Transcription can be evaluated
further by comparison of expression from phage
RNA polymerase promoters versus the endoge-
nous RNA polymerase found in the S30 extract.
Model Enzyme Systems
A number of simple enzymatic assays have
been used in transcription/translation reactions
including -galactosidase, -lactamase, galac-
tokinase, chloramphenicol acetyltransferase,
luciferase, and alkaline phosphatase. -galac-
tosidase is an excellent reporter gene for
screening of in vitro transcription/translation
extracts. Enzyme activity is typically determined
by addition of ONPG, a colorimetric substrate,
which gives measurable readings after a short
incubation.
Luciferase is another common reporter for in
vitro systems. Even very small amounts of func-
tional luciferase can be measured in a
luminescent assay, allowing sensitive detection
of protein synthesis. Both rey and Renilla
luciferase enzymes can be expressed simulta-
neously in the in vitro transcription/translation
reactions and measured using different sub-
strates (Dual-Luciferase
Reporter Assay
Systems
(a,i)
, Cat.# E1910, E1960, E1980). In this
way, both proteins can be expressed and mea-
sured in the same reaction to allow for internal
standardization and controls. This simultaneous
expression can be exploited to immediately
determine whether a compound is acting as an
inhibitor of transcription or translation.
Monitoring and Detection
By far the most common means of monitoring
protein synthesis in the standard analysis of
translation reactions is by the incorporation of
radiolabeled amino acids. Typically,
35
S-labeled
methionine or
14
C-labeled leucine is added to
the reaction. Only newly synthesized proteins
incorporate the radiolabel, allowing specic
detection of the template-encoded polypep-
tides. The radiolabeled proteins can be
visualized by autoradiography after elec-
trophoresis or isolated by immunoprecipitation.
While such detection methods are valuable for
analysis of gene products, they are not readily
adaptable to high-throughput screening meth-
ods. Capture methods using antibodies or other
receptor proteins can be employed, but the dif-
culties in working with and disposing of
radioactivity still remain. Reporter systems wide-
ly used in gene expression offer a more tenable
solution for screening efforts.
The in vitro transcription/translation systems
usually generate proteins that are correctly fold-
ed and retain their enzymatic activity. This
enzymatic activity can be directly assayed as
long as the extract proteins and buffers do not
interfere. The level of synthesis of a reporter
protein is directly dependent on the activity of
the transcription and translation system in the
extract. Therefore, by simply determining the
increased levels of these easily assayed pro-
teins one can determine the relative protein
expression activity of the extract. The individual
transcription and translation components can
be assayed through the choice of template.
For evaluating the effect on translation, mRNA
encoding the reporter gene can be added
directly to the reaction. An example of this
27
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S C R E E N I N G
C H A P T E R F I V E
2. Chen, T.Z. et al. (1997) J. Virol.
Meth. 65, 183.
3. Alt, M. et al. (1997) Arch. Virol.
142, 589.
4. Koppelhus, U. et al. (1997)
5. Azhayeva, E. et al. (1997) Nucl.
6. Curcio, L.D., Bouffard, D.Y. and
Scanlon, K.J. (1997)
Pharmacol.Ther. 74, 317.
7. Lima, W.F. et al. (1997) J. Biol.
Chem. 272, 626.
8. Milner, N., Mir, K.U. and
Southern, E.M. (1997) Nature
Biotech. 15, 537.
9. Kuzchalia, T.V. et al. (1988) Eur.
J. Biochem. 172, 663.
10. Hoeltke, H.J. et al. (1995)
BioTechniques 18, 900.
11. Beckler, G.S. and Hurst, R.
Transcriptional activity can also be measured
directly through the use of a nucleotide analog
that releases a uorophore upon incorporation
into RNA.
Individual reactions require 0.11g of DNA tem-
plate and typically produce 50200ng of protein
from that template in a 3060-minute reaction.
This represents only a small percentage of the
total protein present, and the potential for inter-
fering contaminating activities is always a
concern. Controls should always be employed to
avoid potential problems. The S30 extract is
tolerant of reasonably high levels of organic sol-
vents such as DMF and DMSO, which are
commonly used to dissolve organic compounds.
They are, however, sensitive to alcohols and
these should be avoided as solvents.
In vitro systems are valuable tools for studying
transcriptional and translational machinery. They
are easy to use, and both reactions and assays
are readily adaptable to automation. The reac-
tions and assays are rapid, allowing thousands
of compounds to be evaluated in a screen.
Their in vitro nature makes them more easily
reproducible than in vivo systems. They are also
exible and allow exact additions and combina-
tions of compounds. These properties make
them important additions to the screening
repertoire.
Development of Antiviral Agents
The rapid reticulocyte coupled transcription/
translation screening system has been applied
to the identication of antisense oligodeoxynu-
cleotides capable of inhibiting hepatic D viral
replication (2). Other groups developing specif-
ic viral inhibitors have utilized in vitro expression
to test different target regions with modied
oligonucleotides (peptide nucleic acid and
phosphorothioate oligonucleotides; 3,4).
Additional studies have shown the circular 2-
deoxyribo-oligonucleotides to be potent
inhibitors of luciferase expression in an in vitro
coupled transcription/translation system (5).
Antisense Regulation
Antisense oligonucleotides have been shown to
inhibit gene expression at either the transcrip-
tional or translational level (6). The mechanism
for translational inhibition is thought to involve
activation of RNase and is currently a target of
intense investigation. Attempts to rationally
design effective antisense RNAs have yet to
yield consistent results, giving rise to the use of
an empirical approach using randomized
oligonucleotide arrays to identify ideal candi-
date antisense oligonucleotides (7,8). An in vitro
reticulocyte lysate coupled transcription/transla-
tion system has been established for rapid
screening of antisense oligodeoxyribonu-
cleotides to determine which are the most
effective in arresting mRNA translation (2) The
potential for heteroduplex formation of an
oligonucleotide has been shown to correlate
closely with inhibition of translation in vitro.
Non-Isotopic Detection
Charged tRNA complexes containing epsilon-
modied lysine, such as biotinylated lysines,
can be added to the translation reactions and
the modied lysines will be incorporated into the
synthesized protein. Biotinylated proteins can be
detected non-isotopically using streptavidin-
alkaline phosphatase or horseradish peroxidase
conjugates (9,10); these detection systems
became commercially available in 1993 (11).
28
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12. Gite, S. et al. (2000) Anal.
Biochem. 279, 218.
13. Christopoulos, T.K. and Chiu,
N.H. (1995) Anal. Chem. 67,
4290.
14. Chiu, N.H. and Christopoulos,
T.K. (1996) Anal. Chem. 68,
2304.
15. Laios, E. et al. (2000) Anal.
Chem. 10.1021/ac0004198
(published on the web by the
American Chemical Society).
16. Chiu, N.H.L. and Christopoulos,
T.K. (1999) Clin. Chem.
45,1954.
The same investigators developed a similar
approach for DNA detection (14). This system is
also very sensitive due to the coupled transcrip-
tion/translation system. The resulting
luminescence is linearly related to target DNA
levels for between 5 and 5,000amol. Because
the assay is performed in microtiter wells and
avoids membrane hybridization and blotting, it
can be adapted to automatable HTS detection.
Recently this approach has been expanded to
allow simultaneous detection of the target
sequences using co-expression/determination
of rey and Renilla luciferases (15).
Expression Immunoassay
Most recently work by Chiu and Christopoulos
(16) has extended the use of the coupled in
vitro transcription/translation systems to a
sandwich-type expression immunoassay,
using as a label an expressible DNA fragment
encoding rey luciferase. The DNA label con-
tains a T7 RNA polymerase promoter, a rey
luciferase-encoding sequence and a
poly(dA/dT) tail. The 3-end of the DNA label is
biotinylated and complexed with streptavidin. A
sandwich immunoassay can then be generated
using a coating antibody to a specic antigen,
biotinylated polyclonal Ab to the same antigen
and the streptavidin-luciferase coding DNA
complex. The bound DNA complex label is
expressed in vitro by coupled transcription
and translation.
The luciferase generated is measured using a
standard bioluminescent luciferase assay.
The authors used this technique to success-
fully detect prostate-specic antigen (PSA) in
human serum.
Recently, a method of sensitive nonisotopic uo-
rescence-based detection of nascent proteins
directly in polyacrylamide gels has been report-
ed (12). Nanogram levels of nascent proteins
with incorporated BODIPY
-FL labeled methion-

ine can be detected by laser-based uorescent
gel scanner or even by a conventional UV
transilluminator (12). In addition, Promega
recently introduced a non-isotopic uorescence-
based detection product, the FluoroTect
Green
Lys
in vitro Translation Labeling System
(Cat.# L5001).
Ultra-Sensitive DNA Detection and
Immunoassays
A highly sensitive immunoassay, known as the
ultrasensitive expression immunoassay (USEI)
was developed based on a solid-phase cou-
pled transcription/translation system described
by Christopoulos and Chiu (13). A DNA frag-
ment encoding rey luciferase is biotinylated
and complexed with streptavidin. Biotinylated
specic antibodies are used to quantify antigen
immobilized on microtiter wells. After completion
of the immunoreaction, streptavidin-DNA com-
plex is bound to the immunocomplex. Subse-
quent expression of the solid phase-bound
DNA by a coupled transcription/translation
reaction produces luciferase. As few as 3,000
molecules of DNA label can detect a minimum
of 50,000 antigen molecules. The luminescence
is a linear function of the number of antigen
molecules in a range extending over 3 orders
of magnitude. The high sensitivity achieved
results from the combined amplication due
to transcription/ translation and the substrate
turnover.
R I B O S O M E
D I S P L A Y
6
C H A P T E R
This illustration depicts
antibody-ribosome-mRNA
(ARM) complexes, which
have been used as display
selection particles in an in
vitro eukaryotic method for
selection of antibody-
combining sites. An important
feature of the ARM method is
that it preserves the link
between the peptide of
interest and the gene that
encodes it. The ARM strategy
is based on two ndings: i)
rabbit reticulocyte lysates
(cell-free systems used to
generate proteins) produce
functional single-chain
antibodies; and ii) in a cell-
free system nascent proteins
and their mRNAs form stable
ternary polypeptide-
ribosome-mRNA complexes
in the absence of a stop
codon.
29
30
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Chapter Six: Ribosome Display
Contents Page
Introduction ............................................................................................................ 30
In Vitro Display Libraries ............................................................................................ 31
Introduction
Combinatorial display technologies have proved to be valuable sources of diversity for identifying
ligands to biological molecules. The technologies used are varied but are linked by a common
approach of combining a very large amount of structural diversity with an effective selection to isolate
those rare individuals in the diverse population that interact with the intended target. In vitro
transcription/translation systems offer some unique advantages to this approach.
The most commonly used technique of the display technologies is phage display (13). In phage
display systems, diversity is created through recombinant DNA techniques that introduce a
randomized peptide sequence as a gene fusion to a surface protein on the phage. Each phage is a
separate biological unit that contains both the displayed peptide and the genetic information
encoding the peptide. A physical separation of those phage interacting with the specic target is
employed, typically by interaction on a microtiter plate, and those phage that have bound to the
target are amplied by growth on the appropriate host strain. Following 23 rounds of this enrichment
process, the isolated phage typically bind specically to the target. The identity of the randomized
portion in these clones is determined by conventional DNA sequencing of the obtained phage.
Sequence analysis typically shows a number of related sequences, which can be aligned to
determine a consensus sequence for the binding ligand. Recently in vitro methods have become
more popular because of the potential advantages these approaches hold.
References
1. Smith, G.P. (1985) Science 228,
1315.
2. Burton, D.R. (1995)
Immunotech. 1, 87.
3. Kay, B.K., Winter, J. and
McCafferty, J. (1996) Phage
Display of Peptides and
Proteins, Academic Press, San
Diego.
31
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D I S P L A Y
C H A P T E R S I X
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In Vitro Display Libraries
In vitro display systems incorporate many of the
same principles of the phage systems but have
their own unique characteristics. One of the key
parameters of an effective display system is
having sufcient diversity in the total library of
sequences. In biological systems this is typical-
ly limited by the efciency of transformation of
the initial library of recombinant phage into the
host cell. This practical limitation forces the
diversity of the library to be approximately 10
9
or less. The primary advantage of the in vitro
systems utilizing transcription/translation is over-
coming this limitation in library size by
elimination of the transformation step. In the in
vitro systems utilizing transcription/translation,
DNA template encoding the randomized pep-
tide is added directly to the reaction, and the
diversity is simply a matter of translation ef-
ciency and scale of reaction. Using in vitro
systems, libraries of >10
12
sequences have
been obtained. The increase in library size not
only increases the probability of nding a high-
afnity ligand within the population; it also
increases the number of randomized amino
acid positions that can be completely encom-
passed by the library. The randomized portion
of the displayed peptide is typically encoded by
codons consisting of the sequence NNK where
N represents any nucleotide and K represents
G or T. This degenerate codon then encodes all
of the 20 possible amino acids, as well as a sin-
gle stop codon, through the use of only 32
possible nucleotide combinations rather than
the 64 possible combinations obtained from a
completely random NNN codon. By limiting the
number of codons used, the diversity of amino
acid sequences is increased within the library.
With a phage library of 10
9
sequences, this
diversity represents complete coverage of 6
amino acid positions (32
6
). Using an in vitro
library of 10
12
sequences, this coverage
increases to 8 amino acid positions (32
8
).
Another key property of display libraries that is
a challenge to any in vitro system is the physical
linkage of the randomized peptide to the genet-
ic information that encodes it. In biological
systems this linkage is handled through the
compartmentalization of the genetic material
within the cell and viral particle. In an in vitro
system where the various templates are translat-
ed in a single reaction another way must be
found to link the peptide and the sequence
encoding it. Several approaches to in vitro sys-
tems have been applied to achieve this linkage.
Figure 1. Schematic illustration of ribosome display. The ability to use proteins as the scaffolding structure for the
presentation of the diversity in the libraries has a number of advantages. Scaffolding proteins can provide some
rigidity to the displayed sequence. This rigidity helps to reduce the entropic penalty to binding and tends to
give higher afnity ligands from library panning. Scaffolding proteins can remove the randomized region from
the surface of the ribosome and provide better presentation of that sequence for afnity selection. Additional
functionality can be provided by the scaffolding proteins and for creation of novel combinations of ligands and
effector function. The diversity can also be applied to the activity of the scaffolding protein itself in the search for
variants with novel properties.
DNA
mRNA
mRNA
RT-PCR Translation
Transcription
Isolation
of RNA
Panning
Errors
can be
introduced
Native
protein
tethered
to the
ribosome
5 3
2
6
0
5
M
A
0
3
_
9
A
32
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4. Tawk, D.S. and Grifths, A.D.
(1998) Nature Biotech. 16, 652.
5. Tuerk, C. and Gold, L. (1990)
Science 249, 505.
6. Irvine, D. and Tuerk, C. (1991) J.
Mol. Biol. 222, 739.
7. Vant-Hull, B. et al. (1998) J. Mol.
Biol. 278, 579.
8. Morris, K.N. et al. (1998) Proc.
9. Burke, D.H. et al. (1997) Chem.
Biol. 4, 833.
10. Mattheakis, L.C., Bhatt, R.R.
and Dower, W.J. (1994) Proc.
11. Gersuk, G.M. et al. (1997)
Biochem. Biophys. Res. Comm.
232, 578.
12. Mattheakis, L.C., Dias, J.M. and
Dower, W.J. (1996) Meth.
Enzymol. 267, 195.
13. Borman, S. (1997) Chem. Eng.
News 75, 4.
USA 94, 4937.
15. Roberts, R.W. and Szostak, J.W.
USA 94, 12297.
16. Sikorski, R. and Peters, R.
(1997) Science, 278, 2143.
primary advantages to this approach are the
tremendous diversity that can be created, the
fact that there is no need to link genetic and
structural diversity and the speed at which the
selection process can be performed. The pri-
mary limitation to this approach is the restriction
to the four-base building blocks or a few base
analogs that can be incorporated into the tran-
scribed product.
Another approach that has been applied to in
vitro systems is the use of polysomes for display
(1016; see Figure 1). Polysome display is one
of a number of technologies in the area and
offers some unique advantages. The method
can be performed completely in vitro using a
higher level of diversity than achieved through
biological systems and can be used to express
a variety of protein scaffolds. This method is a
quick and exible alternative to in vivo meth-
ods. Polysomes are the large macromolecular
complexes of ribosomes, mRNA and translated
protein that are formed during the translation
process. Polysome display methods take
advantage of the physical linkage of the genetic
information encoded on the mRNA to the
emerging translation product. The translation in
the polysome complex is arrested either by
addition of an antibiotic such as chlorampheni-
col, by secondary structure in the message, or
by reduction of the reaction temperature. The
One approach is to mimic the in vivo cellular
compartmentalization by creating reverse
micelles, which serve as miniature reaction ves-
sels for the in vitro reaction (4). Transcription/
translation reactions are performed in emulsions
with mineral oil. Individual DNA templates are
contained within the aqueous micelles, and
transcription and translation occurs and is
retained within the aqueous environment of the
micelle. Provided that these micelles can be
effectively screened or selected, they provide a
clonal segregation of the genetic material analo-
gous to an individual phage or cell. The DNA
template can be amplied by PCR
(f)
and multiple
rounds of screening or selection applied to iso-
late those individual clones with the desired
properties.
A second approach to linking the genetic and
structural information within display libraries is
to use the genetic material as the structural
diversity directly. The SELEX approach (59)
creates diversity by creating an RNA library
from a degenerate DNA template. The diversity
of the secondary structure of the transcribed
RNA, as well as the diversity of the primary
sequence of that RNA, are used in the selection
for sequences that bind to the intended target.
Those sequences that bind are identied
through reverse transcription and PCR ampli-
cation followed by DNA sequencing. The
Figure 2. The ARM display cycle. The generation of an ARM library begins with mutagenesis of a V
H
/K template,
followed by antigen-selection of a specic binding ARM and then recovery of the genetic information by RT-
PCR
(f)
.
E. coli
expression
Mutagenesis
T7 V
H
/K
T7
5
...... 3
RT-PCR
Antigen-coupled
magnetic beads
Libraries
(of mutants)
Selection
mRNA
ribosome
in vitro
transcription/
translation
ARM
antibody
fragment
2
1
1
0
M
A
0
3
/
8
A
33
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R I B O S O M E
D I S P L A Y
C H A P T E R S I X
T O O R D E R
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17. He, M. and Taussig, M.J. (1997)
18. Pederson, J. (1998) Promega
Notes 66, 14.
19. Hanes, J. et al. (1998) Proc.
20. Roberts, R.W. (1999) Curr. Opin.
Chem. Biol. 3, 268.
(1999) Curr. Top. Microbiol.
Immunol. 243, 107.
emerging translation product extends from the
ribosome and is available for interaction with
the target protein. After washing to remove
unbound complexes, the complexes are eluted
and the mRNA reverse transcribed and ampli-
ed for further rounds. In addition to the large
diversity that can be screened by this method, it
has the advantage that large proteins can be
used as the scaffold for the diversity.
An example of using polysome display for iso-
lating ligands is shown by the evolution and
selection of antibody combining sites. The
immune response of an animal to a presented
antigen involves a process of selective ampli-
cation of those rare cells producing antibodies
reactive to the antigen. This process can be
copied via a polysome display approach
(17,18,19; see Figure 2). A library of variants
encoding mutations in the antigen combining
sites of single-chain antibodies was expressed
in vitro. Polysomes expressing those variants
were incubated with magnetic beads coated
with the target antigen. Physical separation of
the bound polysomes from unbound was per-
formed and the process repeated for multiple
rounds of enrichment. Using this method,
both the specicity and the afnity of the anti-
body-antigen interaction can be modied.
Enrichments of 10
4
10
5
per round of selection
have been reported with the TNT
T7 Quick
System
(a,b,c,d,e)
(17).
Additional advantages of ribosome display
approaches are that no cloning is required
(PCR
(f)
products are effective substitutes for
T7- or T3- based TNT
Systems) and that exi-

bility is inherent with in vitro approaches. Unlike
phage display, which is limited to an oxidizing
periplasmic environment for protein folding, one
can add components to in vitro systems to
increase functionality, such as chaperonins,
detergents or microsomal membranes. A recent
review of in vitro protein selection approaches is
presented by Roberts (20) and Hanes and
Plckthun (21).
L A R G E S C A L E
P R O T E I N
S Y N T H E S I S
7
C H A P T E R
A number of large-scale
protein synthesis methods
have been developed recently
to increase the yield of cell-
free systems to a preparative
scale. Some of these systems
are prokaryotic and utilize
DNA templates containing
either prokaryotic promoters or
a phage RNA polymerase
promoter and eukaryotic
extracts. Such systems are
utilized to generate micro- to
milligram protein yields per
milliliter of reaction.
34
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L A R G E S C A L E
P R O T E I N
S Y N T H E S I S
C H A P T E R S E V E N
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Chapter Seven: Large-Scale Protein Synthesis
Contents Page
Introduction ............................................................................................................ 35
Improving Yield ........................................................................................................ 36
Continuous-Flow Systems............................................................................................ 37
Modications of the Technique...................................................................................... 38
References
1. Zubay, G. (1974) Annu. Rev.
Genet. 7, 267.
2. Crowley, K.S. et al. (1994) Cell
78, 461.
3. Cornish, V.W. et al. (1994) Proc.
4. Rothchild, K.J. and Gite, S.
(1999) Curr. Opin. Biotech. 10,
64.
5. Gite, S. et al. (2000) Anal.
Biochem. 279, 218.
Introduction
Cell-free systems for in vitro gene expression and protein synthesis have been described for
many different prokaryotic and eukaryotic systems (1). Eukaryotic cell-free systems, such as rabbit
reticulocyte lysate and wheat germ extract, are prepared from crude extract containing all of
the components required for translation of either natural or in vitro transcribed RNA templates.
Prokaryotic systems, however, are typically coupled in that they contain RNA polymerase, which
transcribes mRNA from an exogenous DNA template. During transcription, the 5-end of the mRNA
becomes available for ribosome binding and translation initiation, allowing transcription and transla-
tion to occur simultaneously. Prokaryotic systems are available that utilize DNA templates containing
either prokaryotic promoters (such as lac or tac; E. coli T7 S30 Extract System for Circular DNA
(a,b)
[Cat.# L1130] or a phage RNA polymerase promoter. Coupled eukaryotic cell-free systems have
been developed that combine a prokaryotic phage RNA polymerase/promoter with eukaryotic
extracts and utilize an exogenous DNA template for in vitro protein synthesis (TNT
Coupled
Reticulocyte Lysate
(a,b,c,e)
[Cat.# L4600, L4610, L4950, L5010, L5020] and TNT
Wheat Germ Extract

Systems
(a,b,c,e)
[Cat.# L4380]).
Cell-free expression systems offer several advantages over in vivo expression systems, including the
ability to express toxic, proteolytically sensitive, or unstable gene products. Cell-free systems are
often used to verify that the appropriately sized gene product(s) is synthesized from a cloned gene.
Other applications of in vitro expression systems include analysis of protein-protein and protein-
nucleic acid interactions, mutational analysis, epitope mapping and in vitro evolutionary studies. In
addition, the ability to incorporate unnatural amino acids containing photoactivatable, uorescent,
or biotin groups allows for product analysis by new methods (25).
Typically, standard in vitro systems produce picomole (or nanogram) amounts of proteins per 50l
reaction. This yield is sufcient for most types of analyses, such as polyacrylamide gel separation,
Western blotting, immunoprecipitation and, depending on the protein of interest, enzymatic or bio-
logical activity assays. More recently, a number of methods have been developed to increase this
yield to a preparative scale, which would allow alternative types of analyses and applications for the
in vitro synthesized proteins.
36
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6. Kawarasaki, Y. et al. (1995)
Anal. Biochem. 226, 320.
7. Judice, J.K. et al. (1993)
Science 261, 1578.
8. Wood, K. (1989) Doctoral
Thesis, Univ. of California, San
Diego.
9. Lesley S.A., Brow, M.A.D. and
Burgess, R.R. (1991) J. Biol.
Chem. 266, 2632.
10. Luciferase Assay System
Technical Bulletin #TB281,
Promega Corporation.
Improving Yield
Some investigators have improved the yield
from a standard wheat germ batch translation
system by optimizing the temperature, tRNA
concentration and, most importantly, ATP regen-
eration system (6). These modications
increased yields 4- to 8-fold, resulting in the
synthesis of 30g of protein (E. coli dihydrofo-
late reductase; DHFR) per milliliter of reaction
mixture. Protein synthesis was prolonged for up
to 10 hours with various templates, and both
capped and uncapped mRNA templates were
used. Protein synthesis directed by uncapped
dihydrofolate reductase mRNA containing a viral
cap-independent translation initiation sequence
resulted in the synthesis of 18g DHFR per
milliliter of reaction mixture. The highest yields
were obtained with capped mRNA containing
the viral cap-independent translation initiation
sequence (30g/ml reaction). Protein synthesis
in this improved batch wheat germ system can
sustain a translation reaction for half as long
as the continuous-ow cell free (CFCF) systems
(see below), but it is superior to the CFCF
system in that no special apparatus is required,
and it is more convenient and more repro-
ducible. Large-scale S30 batch reactions syn-
thesizing T4 lysozyme with site-specic incor-
porated spin label (uorescent or photoactiv-
atable amino acids) have produced protein
product yields of 2040g/ml (3,7).
Figure 1. Coomassie
stain detection of SDS-PAGE-separated luciferase gene products synthesized in an E. coli S30 Extract
equilibrium dialysis cell reaction. A 1.5ml E. coli S30 Extract reaction (containing 60g pBESTluc
DNA
(b)
, 600l of
Premix 2.5X Solution, 450l of Promegas E. coli S30 Extract for Circular DNA and 390l of Nuclease-Free Water as
described in reference 8) was separated by a 300kDa MWCO cellulose ester membrane (Amicon, Cat.# XM300)
from a constantly stirred dialysis solution (Premix 1X Solution). The dialysis reaction cell (similar to Fisher Cat.# 08-
666-15), the stir bar in the lower chamber and membrane were sterilized by soaking in 5% formalin for 1 hour,
followed by 3 rinses with distilled water and air-drying in a sterile hood. The reaction proceeded at room temperature
for 20 hours. An identical 1.5ml standard static reaction (1) served as a control reaction. The samples were analyzed
on a 10% SDS-polyacrylamide gel as described (9). Lane 1, 5l of control reaction; lane 2, 5l of control reaction
supernatant (after reaction was centrifuged at 12,000 x g for 5 minutes); lane 3, 5l of dialysis reaction; lane 4, 5l of
dialysis reaction (after reaction was spun at 12,000 x g for 5 minutes); lane 5, puried rey luciferase (1g); lane 6,
2l of Promegas Mid-Range Protein Molecular Weight Markers (no longer available). For determination of functional
luciferase activity, 5l from each reaction were assayed using the Promega Luciferase Assay Reagent
(a)
as
described (10).
66.2
55.0
42.7
40.0
31.0
14.4
1 2 3 4 5 6
Static Reaction
kDa
97.4
Dialysis Reaction Luciferase M
15,019 Luciferase
Activity
(RLU)
Truncated
Luciferase
15,842 36,164 37,143
Truncated
Luciferase
Full-Length
Luciferase
(60.7kDa)
1
3
0
2
G
A
1
2
_
5
A
37
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P R O T E I N
S Y N T H E S I S
C H A P T E R S E V E N
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11. Baranov, V.I. and Spirin, A.S.
(1993) Meth. Enzymol. 217, 123;
U.S. Pat. No. 5,478,730.
12. Kudlicki, W. et al. (1992) Anal.
Biochem. 206, 389.
13. Morozov, I.Y. et al. (1993) Proc.
14. Chetverin, A.B. and Spirin, A.S.
(1995) Prog. Nucl. Acids. Res.
Mol. Biol. 51, 225; U.S. Pat. No.
5,556,769.
in a wheat germ system, 2.0mg globin in a RRL
system and 0.2mg -lactamase or DHFR in an
E. coli S30 system. In general, yield from 1ml of
incubation mixture can vary from 50g to 4mg of
protein depending on the size of the protein, its
solubility, the expressibility of the template and
the type and quality of the cell extract used.
These CFCF systems, however, have several
problems, such as clogging of the ultraltration
membrane, protein aggregation, unexplained
translation disruption, high running cost and low
reproducibility. These problems have been
addressed through additional modications to
the centrifugation steps used when preparing
the E. coli S30 Extract
(a,b)
(12) and the combina-
tion of the Q replicase reaction with the E. coli
S30 Extract System
(a,b)
(13,14). The phage Q
contains an RNA-directed RNA polymerase,
which can efciently amplify RNA in vitro for
large-scale protein synthesis. Signicant stimu-
lation of RNA synthesis by the addition of the
Q replicase enzyme is only observed in the
presence of a completely functioning translation
system. For this type of system, the template of
interest must be cloned into an efcient, natural-
ly occurring Q replicase template (such as
RQ135
-1
RNA). In this system, approximately
0.2pmol DHFR was synthesized per 30l reac-
tion mixture. The continuous production of
sense strand RNA by Q replicase could com-
pensate for the mRNA losses due to
degradation in the CFCF systems, thereby
extending the reaction life and increasing pro-
tein yield.
A disposable dialyzer used in conjunction with
Promegas E. coli S30 Extract System
(a,b)
allowed synthesis of several hundred micro-
grams of protein in a single coupled
transcription/translation reaction. Using dialysis
with this cell-free system can increase protein
synthesis by 10- to 20-fold over standard batch
reactions (180360g luciferase per 1.5ml
reaction; Figure 1). For these experiments, a
DispoDialyzer
(Spectrum
) was used (Figure

2). It is important to select a membrane with a
molecular weight cut-off below the size of the
expressed protein. In addition, the promoter util-
ized, position of ribosome binding site, DNA
template and preparation, reaction temperature
and dialysis solution components may need to
be optimized. In particular, the proper magne-
sium concentration is critical for optimal protein
yields. Such a dialysis system is simple, conve-
nient and economical.
Continuous-Flow Systems
While the static cell-free systems are extremely
useful, they are still limited in the amount of total
protein produced. Spirin and coworkers have
reported several continuous-ow cell-free
(CFCF) systems in which the protein products
are removed through a membrane by pumping
a feeding solution (containing amino acids,
ribonucleotides and energy source) through the
reaction vessel during the course of the 2030-
hour reaction (11). These continuous-ow
systems can produce hundreds of micrograms
of protein from a 1ml reaction. For example, total
protein yields included 0.2mg BMV coat protein
A. B.
1
3
0
5
M
A
1
2
/
5
A
Figure 2. Diagram of the DispoDialyzer
. Panel A: Loading the dialyzer with E. coli S30 Extract. Panel B: The dialysis
reaction consisting of the dialyzer in a sterile 15ml conical tube containing 3.57ml of dialysis solution.
38
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15. Tohda, H. et al. (1994) J.
Biotechnol. 34, 61.
16. Kim, D-M. and Choi, C-Y.
(1996) Biotechnol. Prog. 12,
645.
17. Resto, E. et al. (1992) Nucl.
18. Marszal, E. and Scouten, W.H.
(1996) J. Mol. Recog. 9, 543;
U.S. Pat. No. 6,033,868.
19. Alimov, A.P. et al. (2000)
BioTechniques 28, 338.
Modications of the Technique
Studies by Tohda et al. suggest that increased
protein yield in an E. coli CFCF system may be
obtained using phosphorothioate-containing
mRNA as a template. The thio-mRNA for DHFR
showed higher translational activities than the
corresponding unsubstituted mRNA (probably
due to increased mRNA stability) and the single
substitution of adenosine residues was most
effective in translational activity (15). The thio-
mRNA for DHFR was able to produce the intact
protein possessing catalytic activity.
A reactor for cell-free protein synthesis was
developed by Kim and Choi (16). This system is
similar but simpler than CFCF, in that it enables
extremely high productivity without using any
complex apparatus. The continuous supply of
substrates and removal of by-products was per-
formed by in- and outcome diffusion through a
dialysis membrane, which separates the reac-
tion mixture from the feeding solution. By use of
this system, protein synthesis occurred for at
least 14 hours, yielding 1.2mg chloramphenicol
acetyltransferase (CAT) protein per milliliter of
reaction mixture.
In a GATT (gene amplication with transcription/
translation) system developed by Resto et al.
(17), greater than 10
9
copies of DHFR can be
produced from each plasmid DNA molecule
employed. This system involves sequential cou-
pling of DNA amplication by PCR and in vitro
transcription, followed by in vitro translation in
rabbit reticulocyte lysate. Another group has
reported the use of afnity ligands to continu-
ously remove the synthesized protein products,
thereby improving translation efciency of the
dialysis-based systems (18).
For those laboratories that require intermediate
amounts of protein for analysis, Alimov et al.
(19) recently described a system utilizing E. coli
S30 extract in conjunction with expression vec-
tors that encode viral structural elements known
to enhance translation in vivo and to protect
mRNA from ribonuclease action. The viral ele-
ments include: i) the 133 nucleotide-long cDNA
sequence of an RQ RNA that can be replicated
by Q replicase; and ii) the epsilon ()
sequence, a powerful translational enhancer of
the phage T7 gene coat protein. The designed
vectors also include a Strep-tag oligopeptide at
the C-terminus, which allows afnity purication
of the expressed protein using streptavidin
(K
d
= 10
5
M). The reaction can yield up to
40g/ml, or about 1nmol, of a standard protein.
For the rapid in vitro production of proteins on
the preparative scale, continuous-ow systems
currently have more promise than utility,
although their predictability appears to be
improving. For yields in the hundreds of micro-
gram to low milligram range, the dialysis-based
systems seem most applicable. The develop-
ment of coupled transcription/translation
systems from the hyperthermophilic archaebac-
terial strains may improve CFCF techniques and
allow for more stable and longer lasting systems
than those derived from mesophilic organisms.
P O S T -
T R A N S L A T I O N A L
M O D I F I C A T I O N S
8
C H A P T E R
Cell-free protein synthesis
systems have been observed
to result in a variety of co- and
post-translational protein
modications, such as signal
peptide cleavage or glycosy-
lation (shown here),
phosphorylation, myristoy-
lation and protein folding.
These post-translational
protein modications have
been accomplished in cell-
free systems such as wheat
germ extract and rabbit retic-
ulocyte lysate, both in the
presence and in the absence
of canine pancreatic micro-
somal membranes.
39
40
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Chapter Eight: Post-Translational Modications
Contents Page
Introduction ............................................................................................................ 40
Signal Peptide Cleavage ............................................................................................ 41
Glycosylation .......................................................................................................... 42
Acetylation and Phosphorylation.................................................................................... 42
Isoprenylation and Myristoylation .................................................................................. 43
Protein Folding and Chaperones .................................................................................... 43
Proteolytic Processing and Ubiquitin .............................................................................. 43
Introduction
Several cell-free protein synthesis systems have been used in recent years for the in vitro expression
of proteins from numerous sources. A variety of co- and post-translational protein modications have
been observed in both rabbit reticulocyte lysate (RRL) and wheat germ extract (WGE), the most
commonly used in vitro expression systems, in the presence and absence of canine pancreatic
microsomal membranes. Such modications have been observed in reactions programmed with
template RNA, as well as coupled transcription/translation reactions programmed with DNA.
Modications that have been observed in RRL or WGE include signal peptide cleavage, glycosy-
lation, acetylation, phosphorylation, iso-prenylation, myristoylation, protein folding and proteolytic
processing.
41
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References
1. Creighton, T.E. (1984) Proteins:
Structures and Molecular
Properties, W.H. Freeman and
Company, New York.
2. Walter, P. and Blobel, G. (1983)
Meth. Enzymol. 96, 84.
3. Jackson, R.C. and Blobel, G.
USA 74, 5598.
4. Canine Pancreatic Microsomal
Membranes Technical Manual
5. Bulleid, N.J. et al. (1990)
Biochem. J. 268, 777.
6. Paradis, G. et al. (1987)
Biochem. Cell. Biol. 65, 921.
7. Hirose, S. et al. (1985) J. Biol.
Chem. 260, 16400.
8. MacDonald, M.R. et al. (1988) J.
Biol. Chem. 263, 15176.
9. Bocco, J.L. et al. (1988) Mol.
Biol. Reports 13, 45.
10. Meyer, D.I. (1985) Nature 297,
647.
11. Harnish, D.G. et al. (1986) Mol.
Immunol. 23, 201.
12. Walter, P. et al. (1981) J. Cell.
Biol. 91, 545.
13. Meyer, D.I. (1985) EMBO J. 4,
2031.
14. Powers, T. and Walter, P. (1997)
EMBO J. 16, 4880.
15. Andrews, D. (1987) Promega
Notes 11.
16. Shakin-Eshleman, S.H. et al.
(1996) J. Biol. Chem. 271, 6363.
17. Ghersa, P. et al. (1986) J. Biol.
Chem. 261, 7969.
18. Krebs, H.O. et al. (1989) Eur. J.
Biochem. 181, 323.
19. Scheele, G. (1983) Meth.
Enzymol. 96, 94.
20. Morimoto, T. et al. (1983) Meth.
Enzymol. 96, 121.
21. Spiess, M. et al. (1989) J. Biol.
Chem. 264, 19117.
Signal Peptide Cleavage
The most commonly investigated co- and post-
translational modications to in vitro synthesized
proteins are signal peptide cleavage and N-
linked glycosylation. Proteins destined for
secretion, membrane insertion or inclusion into
the lumen of certain cellular organelles contain
a characteristic sequence at their N-terminus,
designated the signal sequence. The signal
sequence interacts with the signal recognition
particle (SRP), which targets the translating pro-
tein to the rough endoplasmic reticulum (RER)
by interacting with the SRP receptor (docking
protein). Upon insertion into the RER, the signal
sequence is removed by a specic protease,
termed signal peptidase (1).
Signal peptide cleavage may occur with the
appropriate template in rabbit reticulocyte lysate
(RRL) in the presence of canine pancreatic
microsomal membranes (2), as the proteins are
translocated into the microsomal membrane
interior. The enzyme responsible for signal pep-
tide cleavage (signal peptidase) has been
puried from canine pancreatic microsomal
membranes (3). Signal peptide processing,
commonly observed as a shift to a lower
molecular weight upon analysis with SDS-poly-
acrylamide gel electrophoresis, has been
observed with a number of different protein sub-
strates, including E. coli -lactamase (4; Figure
1, Panel A), human interferon- (5), human pro-
static acid phosphatase (6), human renin (7)
and -, - and -preprotachykinins (8). Signal
peptide cleavage has not been detected in RRL
in the absence of microsomal membranes
(48). This more rapidly migrating band arising
from signal peptide processing would also be
predicted to be protected from exogenous pro-
tease degradation (see below).
Signal peptide cleavage has also been detect-
ed in WGE supplemented with canine
microsomal membranes with in vitro synthe-
sized proteins such as human placental SP
1
(9),
preprolactin and pregrowth hormone (3) and -,
- and -preprotachykinins (8). Studies investi-
gating the signal peptide processing of the
preprotachykinins demonstrate that, at least for
this particular protein substrate, RRL was much
more efcient in processing compared to WGE
(8). No signal peptide cleavage has been
detected in WGE in the absence of microsomal
membranes (3). Please note that Promegas
Wheat Germ Extract has been processed for
maximal translation. During this process the
SRP is removed, thus Promegas WGE will not
function with CMMs. Studies by a number of
laboratories have demonstrated that WGE lacks
the SRP found in RRL (1012), and that addition
of SRP to WGE relieves the translation block
that can occur with certain templates in WGE in
the presence of microsomal membranes
(1013). This translation block generally occurs
with secretory or membrane proteins and is not
observed with the in vitro expression of cyto-
plasmic proteins in this system (12). Interestingly,
SRP and SRP receptor (docking protein)
homologs have been identied in bacteria,
and these homologs are functional in the RRL
system (14).
Protein translocation into the interior of mem-
branes is often conrmed using protease
protection assays, in which the translation reac-
tion is incubated with a protease such as
proteinase K (5,1518) or trypsin (2,8,1922).
Figure 1. Processing and glycosylation activity of Canine Pancreatic Microsomal Membranes. The positive control
mRNAs (Panel A, 0.1g of E. coli -lactamase; Panel B, 0.1g of S. cerevisiae -factor) were translated using
Promegas Rabbit Reticulocyte Lysate in a 25l reaction for 60 minutes in the pres-ence of the indicated
amounts of Canine Microsomal Membranes
(c)
(CMM) using [
35
S]met. Aliquots (1l ) were analyzed by SDS-
PAGE on a 420% Novex
gel, transferred to a sheet of PVDF (Bio-Rad
, Sequi-Blot) and exposed to a

phosphorimaging cassette plate for 12 hours.
precursor
(~31kDa)
processed
(~28kDa)
A.
l
CMM
B.
processed
(~30kDa)
precursor
(~18kDa)
0 0.3 0.6 0.9 1.2 1.5 1.8 2.1 2.4
0 0.3 0.6 0.9 1.2 1.5 1.8 2.1 2.4
l
CMM
2
9
2
3
T
A
0
4
/
3
B
42
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22. Miao, G-H. et al. (1992) J. Cell.
Biol. 118, 481.
23. Cauleld, M.P. et al. (1986) J.
Biol. Chem. 261, 10953.
24. Zhou, X. et al. (1993) Biochem.
Biophys. Res. Comm. 192,
1453.
25. Zhang, J-T. and Ling, V. (1995)
Biochem. 34, 9159.
Biochem. J. 286, 275.
27. Hille, A. et al. (1989) J. Biol.
Chem. 264, 13460.
28. Marquardt, T. et al. (1993) J.
Biol. Chem. 268, 19618.
29. Chen, M. and Zhang, J-T.
(1996) Mol. Memb. Biol. 13, 33.
30. Rubenstein, J.L.R. and
Chappell, T.G. (1983) J. Cell.
Biol. 96, 1464.
31. Lopez, C.D. et al. (1990)
Science 248, 226.
32. Starr, C.M. and Hanover, J.A.
(1990) J. Biol. Chem. 265,
6868.
33. Kottler, M.L. et al. (1989) J.
Steroid. Biochem. 33, 201.
34. Palmiter, R.D. et al. (1978) Proc.
35. Rubenstein, P. et al. (1981) J.
Biol. Chem. 256, 8149.
36. Jackson, R. and Hunter, T.
(1970) Nature 227, 672.
37. Palmiter, R.D. et al. (1977) J.
Biol. Chem. 252, 6386.
N-linked glycosylation is generally detected
by the shift to a higher molecular weight form
upon analysis by SDS-polyacrylamide gel
electrophoresis. The presence of N-linked gly-
cosylation is then conrmed by digestion of the
expressed protein with either endoglycosidase
H (6,9,15,21,22), N-glycanase (5), glycopepti-
dase A (7), or -mannosidase (9) prior to gel
electrophoresis. After disruption of the lipid
bilayer of the membranes with 0.1% of the non-
ionic detergent Nikkol, core glycosylation does
not occur, although signal peptide cleavage
remains intact, allowing for the discrimination
between these two processing events (2,28).
Glycosylation can also be inhibited by the addi-
tion of N-benzoyl-Asn-Leu-Thr-N-methylamide
to the translation reaction (27).
N-linked glycosylation has also been detected
in WGE supplemented with canine microsomal
membranes with various in vitro expressed sub-
strate proteins (18,25,30). Studies by Spiess et
al. (21) indicated that different stop-transfer sig-
nals for translocation into microsomal
membranes may be utilized with different ef-
ciencies in RRL compared to WGE. In addition,
studies by Zhang and Ling (25) suggested that
the membrane topology of expressed proteins
in the presence of microsomal membranes is
different depending on the in vitro expression
system utilized (RRL versus WGE). Studies by
Lopez et al. (31) suggested that RRL contains a
cytoplasmic component necessary for the gen-
eration of secretory forms for certain proteins,
which is not present in WGE.
O-linked glycosylation has been observed in the
absence of microsomal membranes in RRL,
while this same modication was not observed
with the same template in WGE (32). The addi-
tion of O-linked oligosaccharides probably
occurs post- translationally, as opposed to the
co-translational addition of N-linked oligosac-
charides (32). The lack of glycosylation in WGE
in the absence of microsomal membranes was
also observed by Kottler et al. (33).
Acetylation and Phosphorylation
In addition to signal peptide cleavage and N-
linked glycosylation, other co- and post-
translational protein modications have been
observed in these in vitro expression systems.
The removal of the N-terminal methionine
residue has been observed in RRL for various
protein substrates including ovalbumin and oth-
ers (3437), and N-Met removal is sometimes
followed by N-terminal acetylation (34). N-termi-
nal acetylation is usually produced early in
translation when the nascent chain is still
attached to the ribosome, but little is known
The protease reactions are performed in the
presence or absence of a nonionic detergent,
such as Triton
X-100 (2,8,17,18,22) or Nonidet
P-40 (21), because protection due to microso-

mal incorporation only occurs in the absence of
detergent. Translocation may also be detected
by separation of the intact membranes from the
translation reaction using ultracentrifugation, fol-
lowed by the subsequent detection of the
translocated or intramembrane proteins in the
membrane pellet (19). Protease protection of
human placental lactogen after post-translation-
al insertion into microsomal membranes was
observed by Cauleld et al. (23). In addition,
studies by Miao et al. (22) suggested that
cotranslational insertion into microsomal mem-
branes can occur in the absence of signal
peptide cleavage.
Glycosylation
Glycoproteins are generated by the addition of
oligosaccharides to the NH
2
group of
asparagine (N-linked) or to the OH group of ser-
ine, threonine, or hydroxylysine (O-linked
glycosylation). N-linked glycosylation initiates in
the lumen of the RER and further processing
occurs in the Golgi apparatus. O-linked glyco-
sylation occurs in the Golgi apparatus (1).
N-linked glycosylation has been detected with a
number of different templates when expressed
in RRL in the presence of canine microsomal
membranes. These include S. cerevisiae -mat-
ing factor (4; Figure 1, Panel B), human
interferon- (5), human prostatic acid phos-
phatase (6), human renin (7), rabies virus
glycoprotein (16), pro-sucrase-isomaltase (17),
asialoglycoprotein receptor H1 (21), human
insulin proreceptor (24), P-glycoprotein (25), tis-
sue-type plasminogen activator (TPA; 26),
mannose 6-phosphate receptor (MPR; 27) and
inuenza hemagglutinin (28). Translation reac-
tions for TPA (26), MPR (27) and inuenza
hemagglutinin (28) were performed in the pres-
ence of reduced glutathione (GSSG) to allow for
correct glycosylation and protein folding, and
thus native enzymatic activity. In vitro expression
of the cystic brosis transmembrane conduc-
tance regulator (CFTR) in RRL in the presence
of microsomal membranes showed the CFTR to
be glycosylated in the absence of signal pep-
tide cleavage and not glycosylated when
translated in WGE in the presence of microso-
mal membranes (29). The lack of glycosylation
of the CFTR protein in WGE was postulated to
be due to the lack of SRP in WGE, and expres-
sion of CFTR in RRL in the absence of
membranes required the presence of Triton
X-
100 to release the SRP block (29).
43
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P O S T -
C H A P T E R E I G H T
T O O R D E R
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38. Kasten-Jolly, J. and Abraham
E.C. (1986) Biochim. Biophys.
Acta. 866, 125.
39. Palmiter, R.D. (1983) Meth.
Enzymol. 96, 150.
40. Gibbs, P.E.M. et al. (1985)
Biochim. Biophys. Acta 824,
247.
41. Curran T. et al. (1987)
Oncogene 2, 79.
42. Prochownik, E.V. and
VanAntwerp, M.E. (1993) Proc.
43. Schubert, U. et al. (1994) J. Mol.
Biol. 236, 16.
44. Joshi, B. et al. (1995) J. Biol.
Chem. 270, 14597.
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Biol. Chem. 254, 762.
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Chem. 268, 11670.
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48. Newman, P. et al. (1991)
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227.
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53. Hancock, J.F. (1995) Meth.
Enzymol. 255, 60.
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Acad. Sci. USA 86, 6858.
55. Li, J. and Aderem, A. (1992)
Cell 70, 791.
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Cell. Biol. 8, 4295.
57. Chow, M. et al. (1987) Nature
327, 482.
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about its signicance (1). N-terminal acetylation
can also occur in the absence of the removal of
the N-terminal methionine residue (35,3840).
Acetylation can be inhibited by the addition of
S-acetonyl CoA to the translation reaction (35).
The addition of phosphate groups to serine,
threonine or tyrosine residues in cellular proteins
is a common post-translational modication,
and for many proteins this modication alters
their biological activity or function. Phospho-
rylation is mediated by the action of a wide
variety of protein kinases present in the cellular
milieu (1).
Phosphorylation of in vitro expressed proteins
has also been observed. Examples of proteins
phosphorylated in RRL include human type I
keratins (40), rat c-fos (41), max proteins (42),
human immunodeciency virus type I vpu pro-
tein (43) and eIF-4E (44). Phosphorylation of the
type I keratins appeared to occur post-transla-
tionally, following release from ribosomes (40).
Phosphorylation events are likely due to cAMP-
dependent protein kinase (PKA; 41), protein
kinase C (PKC; 44), casein kinase II (42,43,45)
and potentially other kinases as well. The ser-
ine/threonine phosphorylation of the max
proteins could be inhibited by staurosporine
(42). Phosphorylation in WGE and RRL has
been demonstrated for in vitro expressed chick-
en myoD1 (46). The addition of 3mM EDTA to
the reaction blocked the phosphorylation of
myoD1 (46).
Isoprenylation and Myristoylation
The linkage of isoprenoid (lipid) groups to
cysteine residues represents an important type
of post-translational protein modication in
eukaryotic cells and may play a role in mem-
brane localization (47). Isoprenylation of in vitro
expressed proteins translated in the RRL sys-
tem have also been demonstrated. Isoprenylation
was observed for human p11 protein (48), the
G-protein subunit (47,49), Rap2a and Rap2b
(50), the Hepatitis delta virus large antigen (51)
and the human homolog of bacterial DnaJ (52).
The lipid moieties attached include both farne-
syl (49,50,52) and geranylgeranyl groups
(4951). Farnesylation may be inhibited by the
addition of -hydroxy-farnesylphosphonic acid
to the translation reaction (52). In the presence
of microsomal membranes, isoprenylation, as
well as methylation and AAX endopeptidase
cleavage, have also been demonstrated in RRL
for the ras superfamily of GTPases (53). In addi-
tion, the glypiation of protein substrates has
been demonstrated for mouse Thy-1 antigen
and human decay-accelerating factor when
expressed in RRL supplemented with microso-
mal membranes (54). Glypiation involves the
addition of glycosyl-inositolphospholipid struc-
tures to the C-terminus of proteins (54).
Proteins expressed in vitro using the RRL sys-
tem may also be modied by myristoylation
(49,5558). The linkage of myristoylate groups
to proteins may play a role in membrane local-
ization, similar to isoprenylation (47). Using
synthetic octapeptide substrates, N-myristoyl-
transferase (NMT) activity was detected in both
RRL and WGE (58). Myristoylation was also
detected when the vaccinia virus gene encod-
ing the major late myristoylated virion protein
L1R was expressed in WGE (59). Myristoylation
of the L1R protein in the WGE also involved
removal of the N-terminal methionine residue
(59).
Protein Folding and Chaperones
Protein folding and renaturation in vivo are often
mediated by cellular chaperones, present both
in the cytoplasm and endoplasmic reticulum of
cells (see reference 60 for a review).
Chaperones generally interact with hydrophobic
regions of newly synthesized or denatured pro-
teins to prevent aggregation or spurious
interactions with other proteins. Molecular chap-
erones and chaperone activity have been
detected in RRL and canine pancreatic micro-
somal membranes and include Hsp90
(61,6367), Hsp70 (6163,65,66), BiP (67), pro-
tein disulde isomerase (67), p60 (61), p48
(61), p23 (61,63), calnexin (67), Hop (63), Hip
(63), Hsp 56 immunophilin (65), Hsp40 (66) and
TriC (66). Frydman et al. (66) quantitated the
levels of Hsp70, Hsp90 and TriC in RRL to be
2M, 2M and 0.7M, respectively. Functional
Hsp90 has not been detected in WGE (68,69),
and differential folding has been observed with
various protein substrates when expressed in
RRL and WGE, resulting in active protein only in
the RRL (6870). The correct folding for in vitro
expressed mutant p53 was demonstrated to
require functional Hsp90 (68). Similarly, func-
tional Hsp90 was required for the in vitro
expression of functional glucocorticoid receptor
(69). The in vitro synthesis of an active protein
in some instances requires the addition of oxi-
dized glutathione to RRL (2628,71).
Proteolytic Processing and Ubiquitin
An additional type of co- or post-translational
modication, which may occur during in vitro
expression, is proteolytic processing. Rabbit
reticulocyte lysate has been shown by a num-
ber of laboratories to contain an active ubiquitin
conjugation and degradation pathway, includ-
ing active 26S and 20S proteosome systems
(7280). In general, small proteins (less than
15kDa) expressed in RRL may be particularly
44
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R.L. (1996) Biochem. 3555,
13443.
62. Hainaut, P. and Milner, J. (1992)
EMBO J. 11, 3513.
63. Johnson, B.D. et al. (1998) J.
Biol. Chem. 273, 3679.
64. Hartson, S.D. et al. (1998) J.
Biol. Chem. 273, 8475.
65. Hutchison, K.A. et al. (1994) J.
Biol. Chem. 269, 5043.
66. Frydman, J. et al. (1994) Nature
370, 111.
67. Roy, S. et al. (1996) J. Biol.
Chem. 271, 24544.
68. Blagosklonny, M.V. et al. (1996)
Proc. Natl. Acad. Sci. USA 93,
n8379.
69. Dalman, F.C. et al. (1990) J.
Biol. Chem. 265, 3615.
70. Lain, B. et al. (1994 ) J. Biol.
Chem. 269, 15588.
71. Omura, F. et al. (1991) Eur. J.
Biochem. 198, 477.
72. Tamura, T. et al. (1991) FEBS
Lett. 292, 154.
73. Wajnberg, E.F. and Fagan, J.M.
(1989) FEBS Lett. 243, 141.
74. Bercovich, Z. and Kahana, C.
(1993) Eur. J. Biochem. 213,
205.
75. Tibbles, K.W. et al. (1995) J.
Gen. Virol. 76, 3059.
76. Mori. S. et al. (1997) Eur. J.
Biochem. 247, 1190.
77. Hoffman, L. et al. (1992) J. Biol.
Chem. 267, 22362.
78. Gonen, H. et al. (1991) J. Biol.
Chem. 266, 19221.
79. Hough, R. et al. (1987) J. Biol.
Chem. 262, 8303.
80. Sato, S. et al. (1998) J. Biol.
Chem. 273, 7189.
81. Nielsen, K.H. et al. (1997) Mol.
Cell. Biol. 17, 7132.
82. Fagan, J.M. et al. (1986) J. Biol.
Chem. 261, 5705.
83. Zagorski, W. et al. (1983)
Biochimie 65, 127.
84. Mumford, R.A. et al. (1981)
Biochem. Biophys. Res. Comm.
103, 565.
activities, while in addition, WGE also possess-
es trypsin-like and post-proline cleavage
activities (83,84). Thus the stability of an in vitro
expressed protein can vary depending on the
type of expression system used and specic
characteristics of the expressed protein.
Rabbit reticulocyte lysate, wheat germ extract
and canine pancreatic microsomal membranes
are complex biological systems, and thus the
various co- and post-translational modications
observed in in vitro expression reactions utiliz-
ing these systems may vary among different
preparations of lysate, extract, or microsomes.
The protein product of an in vitro expression
reaction may deviate from the predicted molec-
ular weight due to one or more of the many
possible modications discussed here, as well
as other uncharacterized modications. Only by
further experimental investigation can the nature
of the modications, as well as their potential
functional and physiological signicance, be
determined.
susceptible to degradation by the ubiquitin
pathway, and WGE may be a more suitable sys-
tem for the in vitro expression of small proteins.
The preferential degradation of in vitro
expressed ornithine decarboxylase (74) and
coronavirus infectious bronchitis virus 1a
polyprotein (75) have been demonstrated in
RRL. Studies by Wajnberg and Fagan (73) and
Sato et al. (80) have shown that hemin and
polyamines can inhibit the ubiquitin proteosome
degradation pathway, and in general, commer-
cial preparations of RRL contain exogenously
added hemin. The addition of ATPS to the
translation reaction can inhibit degradation with-
out interfering with ubiquitination (81). Protein
degradation pathways independent of ubiquitin
and ATP have been identied in RRL as well
and appear to target oxidatively damaged pro-
teins for proteolysis (82). In addition, the
differential degradation of Sos1 versus mSos2
has been observed in RRL but not in WGE (81).
Both RRL and WGE appear to contain amino-
peptidase-, chymotrypsin- and elastase-like
P R O T E I N
F U N C T I O N
9
C H A P T E R
Proteins produced in vitro
in cell-free systems exhibit
varying degrees of protein
function or activity,
depending on the factors
necessary for correct
synthesis, folding and
cofactor incorporation. In
this illustration the protein
demonstrates lumines-
cence once the essential
cofactor ATP is present.
45
46
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Chapter Nine: Protein Function
Contents Page
Introduction ............................................................................................................ 46
Enzymatic Activity .................................................................................................... 47
Other Functions ........................................................................................................ 47
Introduction
Various expression systems for the in vitro production of exogenous proteins are currently in use. The
proteins expressed in the coupled and uncoupled rabbit reticulocyte lysate (RRL), wheat germ
extract (WGE), and E. coli S30 Systems include both prokaryotic and eukaryotic templates, and
exhibit varying degrees of activity upon completion of translation. The level of activity of a particular
protein when expressed in an in vitro expression system will be dependent on the factors necessary
for correct synthesis, folding, and cofactor incorporation if necessary, and whether those factors are
present in the in vitro expression system being used.
Many different types of proteins have been expressed in vitro, and the activity of these pro-teins has
been determined using many different enzymatic and functional assays. Most commonly, RNA- or
DNA-binding proteins are expressed in vitro, and the activity of such proteins determined using
mobility (gel) shift assays with the appropriate RNA or DNA probe. For such studies, the choice of in
vitro expression system can be critical, as back-ground RNA or DNA binding activities can vary
dramatically between rabbit reticulocyte lysate, wheat germ extract and E. coli S30. For more detailed
information concerning the in vitro expression and functional determination of nucleic acid-binding
proteins, see Chapter 3 of this guide.
Another common function of in vitro expressed proteins is the determination of protein-protein interac-
tions. Proteins expressed by in vitro systems may be assayed for the ability to bind to other proteins
endogenous to the expression system or to exogenous proteins either supplied as pure or semi-pure
proteins. Alternately, the target protein may also be expressed in vitro, in either the same reaction or in
a separate reaction as the bait protein. For a more detailed discussion concerning the determination
of protein-protein interactions with in vitro expressed templates, see Chapter 2 of this guide.
The functionality of other in vitro expressed proteins has been assayed using a number of different
techniques, and the remainder of this chapter will provide some examples of pro-teins and activities
detected following in vitro expression.
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References
1. Rabbit Reticulocyte Lysate
System Technical Manual
2. TNT
Coupled Reticulocyte
Lysate Systems Technical
Bulletin #TB126, Promega
Corporation.
3. Wheat Germ Extract Technical
Corporation.
4. TNT
Wheat Germ Extract

Systems Technical Bulletin
#TB165, Promega Corporation.
5. E. coli S30 Extract System for
Circular DNA Technical Bulletin
6. E. coli S30 System for Linear
Templates Technical Bulletin
7. Schneider, C. et al. (1996) Proc.
R.L. (1996) Biochem. 35,
13443.
9. Sung, D. and Kang, H. (1998)
Photochem. Photobiol. 68, 749.
10. Omura, F. et al. (1991) Eur. J.
Biochem. 198, 477.
11. Hemmer, O. et al. (1995)
Virology 206, 362.
12. Pieroni, L. et al. (1997) J. Virol.
71, 6373.
13. Godefroy, S. and Guenet, C.
(1995) FEBS Lett. 357, 168.
Biochem. J. 286, 275.
15. Stoltz, M. et al. (1995) J. Biol.
Chem. 270, 8016.
16. Lain, B. et al. (1994) J. Biol.
Chem. 269, 15588.
17. Murthy, M.S.R. and Pande, S.V.
(1994) Biochem. J. 304, 31.
18. Fessing, M.Y. et al. (1998)
FEBS Lett. 424, 143.
19. Shin, D-Y. et al. (1997)
Oncogene 14, 2633.
20. Mikulits, W. et al. (1997)
Biochim. Biophys. ACTA 1338,
267.
21. Endo, Y. et al. (1992) J.
Biotechnol. 15, 221.
22. Ghosh, A. et al. (1997) J. Biol.
Chem. 272, 33220.
Enzymatic Activity
The expression and enzymatic activity of rey
luciferase are commonly used as both positive
controls for in vitro expression system functions
(16), and for studies investigating protein
refolding in vitro (7,8) or protein characterization
through mutagenesis studies (9).
Other enzymatic functions for in vitro expressed
proteins have also been measured in addition to
luciferase, and include protease, transferase,
phosphatase, kinase, reductase, synthase and
oxidase activities. Functional human lysozyme
has been expressed in RRL (10), while a num-
ber of active viral proteases, including those
from tomato black ring virus (TBRV), hepatitis C
(HCV), and herpes simplex virus (HSV-1), have
been expressed in either RRL or WGE (1113).
Active tissue-type plasminogen activator (TPA)
has been expressed in RRL in the presence of
canine microsomal membranes and oxidized
glutathione (14), while active 6-hydroxy-D-nico-
tine oxidase was expressed in RRL supple-
mented with the cofactor FAD (15). A variety of
active transferase enzymes have been success-
fully expressed and detected in either RRL or
WGE, and include aspartate aminotransferase
(16), carnitine palmitoyltransferase (17), and
thiopurine S-methyltransferase (18). Expression
of B59 ERK phosphatase (19) and murine
thymidine kinase (20) in RRL produced func-
tional proteins with detectable phosphatase and
kinase activities, respectively. A continuous-ow
cell-free WGE translation system was used to
synthesize active dihydrofolate reductase (21),
while RRL has been utilized to express function-
al 25-oligoadenylate synthase (22). Human
hepatitis B virus (HBV) polymerase was
expressed by RRL-coupled transcription/trans-
lation. The in vitro expressed polymerase
possessed protein priming activity demonstrat-
ed by [
32
P]-dGTP labeling. In addition,
polymerization activity was evident by synthesis
of HBV-specic DNA products between 100
and 500 nucleotides. Polymerization activity was
also detected in in vitro polymerase assay by
incorporation of radionucleotides into acid pre-
cipitable polynucleotides (23).
Proteins with incorporated biotinylated lysines
can also be assayed for enzymatic activity. For
example, pectin methylesterase was assayed
enzymatically from TNT
and Transcend
Systems (24).
Other Functions
Biologically active proteins have been suc-
cess-fully expressed in vitro. Active human
interleukin-6 (IL-6) has been synthesized using
a continuous-ow cell-free WGE translation
system (26), as has interleukin-2 (IL-2) (27).
The functional assembly of protein subunits into
a mature, active complex has been demonstrat-
ed for brinogen (28), connexins (29) and
steroid hormone receptors (30). The expression
of functionally active complexes requires the
presence of canine microsomal membranes for
both brinogen and connexins, and the activity
of functional brinogen also requires the pres-
ence of oxidized glutathione during translation.
The assembly of connexin proteins into function-
al gap junction channels was demonstrated
using single channel conductance for connex-
48
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23. Li, Z. and Tyrrell, D.L. (1999)
Biochem. Cell Biol. 77, 119.
24. Wen, F., Zhu, Y. and Hawes,
M.C. (1999) Plant Cell 11, 1129.
25. Beckler, G. S. and Hurst, R.
26. Volyanik, E.V. et al. (1993) Anal.
Biochem. 214, 289.
27. Kolosov, M.I. et al. (1992)
Biotechnol. Applied Biochem.
16, 125.
28. Roy, S. et al. (1996) J. Biol.
Chem. 271, 24544.
29. Falk, M.M. et al. (1997) EMBO
J. 16, 2703.
30. Dittmar, K.D. et al. (1996) J.
Biol. Chem. 271, 12833.
31. Hille, A. et al. (1989) J. Biol.
Chem. 264, 13460.
32. Edelmann, A. et al. (2000) Eur.
J. Biochem. 267, 4825.
33. Spearman, P. and Ratner, L.
(1996) J. Virol. 70, 8187.
34. Iyengar, S. et al. (1996) Clin.
Diagnost. Lab. Immunol. 3,
733.
35. Joyce, G.F. (1993) Pure Appl.
Chem. 65, 1205.
like particles that closely resembled papillo-
mavirus virions and that retained various
conformational epitopes.
More recently, the use of in vitro transcription/
translation systems for in vitro expression
cloning (IVEC
(k)
) has become more widely used
as an alternative to library screening with either
nucleic acid or antibody probes. This method of
cloning is discussed in more detail in Chapter 6.
Finally, the use of coupled in vitro transcription/
translation systems may be used in the future for
the in vitro evolution of catalytic function (35).
Laboratory evolution has been carried out suc-
cessfully with RNA molecules, and studies by
Joyce and coworkers were able to operate
isothermal RNA amplication and in vitro trans-
lation simultaneously with the TNT
RRL
System
(a,b,c,e)
. The problem of colocalization of
gene and protein product remains for this type
of in vitro evolution system.
Thus a wide variety of proteins can be
expressed in their active states in vitro, and
these activities can be measured using many
different types of assays and techniques. The
success of expressing and detecting a specic
protein will depend highly on the efciency with
which the particular protein of interest is trans-
lated and folded in the in vitro expression
system used, as well as the potential back-
ground activities that may be present in the
expression system itself. For many proteins, this
information can only be obtained empirically by
experimentation and optimization of both protein
expression and functional detection.
ins that were integrated into microsomal vesi-
cles. Rabbit reticulocyte lysate contains a
multiprotein system that assembles steroid
hormone receptors and, in particular, the gluco-
corticoid receptor, into an active heterocomplex
with heat shock protein 90 (hsp90).
The addition of oxidized glutathione to in vitro
expression systems to allow for the synthesis
of active proteins is not uncommon and is nec-
essary for the expression of active mannose-
6- phosphate-specic receptor (30). In this
instance, protein activity was determined using
phosphomannan afnity chromatography.
PFK1 and PFK2 coding for the subunits of 6-
phosphofructokinase were cloned into plasmids.
In vitro translation products resulted, using RRL
as the synthesis and folding system. Folding
and assembly of both the - and -subunits of
6-phosphofructokinase occurred, resulting in an
enzymatically active protein. The in vitro-gener-
ated enzyme exhibited a folding state similar to
that of the heterooctameric 6-phosphofructoki-
nase, as demonstrated by size exclusion
followed by ELISA (32).
The assembly of intact viral capsids in vitro has
been demonstrated for a number of viral coat
proteins, including human immunodeciency
virus type 1 (HIV-1; 33) and human papillo-
mavirus type 16 (HPV-16; 34). Rabbit reticulocyte
lysate in vitro translation was used to create HIV
capsids in vitro, and these in vitro capsids
appeared essentially identical to immature cap-
sids produced in vivo as demonstrated by
electron microscopy. Expression of the HPV L1
protein in RRL resulted in the assembly of virus-
49
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Bridging the Gap Between Genomics and Proteomics: In Vitro Expression Tools for

Systems). Introduced in 1992 and continually improved, the TNT

Systems have been used to rapidly screen

Systems supplemented with

Systems (5). This schematic shows translation of one

System reaction. Large amounts of the suspected partner

reaction. The agarose is

beads. After the beads were washed, retained FADD

reactions expressing either

Wheat Germ System.

Wheat Germ Extract

Coupled Reticulocyte Lysate

Systems for studying protein-DNA and protein-RNA interactions. An unlabeled (non-

System reaction and then mixed with a

reaction and added directly to the assay.

Soft Release Avidin

Biotinylated tRNA, Cat.# L5061) in a

System programmed with plasmid

Wheat Germ System. c-Rel, p98 and

Wheat Germ Extract

reaction products added to each

Coupled Transcription/Translation System

tRNA (12) and were bound to 100l of SoftLink

Soft Release Avidin Resin

T7 Quick for PCR DNA

-FL labeled methion-

Systems) and that exi-

Wheat Germ Extract

) was used (Figure

gel, transferred to a sheet of PVDF (Bio-Rad

, Sequi-Blot) and exposed to a

X-100 (2,8,17,18,22) or Nonidet

P-40 (21), because protection due to microso-

Wheat Germ Extract

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