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Developments in recombinant DNA technology have provided an alternate route for the production of proteins.
Gene expression and production of proteins of interest are of great importance for basic research as well as for
biomedical applications. A number of expression systems using mammalian cells, insect cells, yeast and other
bacteria as host have been developed. Yeast has received attention as a suitable host for expression of many
mammalian genes due to many specific characteristics. Yeast strains, Schizosaccharomyces pombe, Saccharomyces
cerevisiae and Pichia pastoris, have many advantages over other systems and may be the host of choice for the
expression of complex proteins of therapeutic value. During the post-genomicera, the importance of these strains for
the expression of heterologous genes may enhance considerably.
Keywords: Schizosaceharomyces
Introduction
Antibodies have been used to identify, localize,
quantitate and analyze proteins. Before gene cloning,
only the natural sources were available for the
isolation of proteins, to which antibodies could be
raised. Low protein yields and the associated
impurities often compromised the quality and quantity
of antibodies.
However,
recent
advances
in
recombinant DNA technology have provided an
alternate route for the production of many proteins of
biomedical importance. The expression of foreign
genes and production of proteins of interest are very
important for both the basic research such as
elucidation of physiological activity, its modulation,
analysis of structure-function relationship of control
elements and regulation of gene expression as well as
practical applications
related to production of
pharmaceuticals. The demand for expression systems
suitable for high-level synthesis of foreign gene
products is, therefore, increasing rapidly.
The expression systems consist combinations of
various genetic elements of host and vector. A
number
of expression
systems,
some using
prokaryotes like Escherichia coli, Bacillus spp.,
Streptomyces
spp. as host and others using
Aspergillus, yeast, insect cells, mammalian cells and
other eukaryotes, have been developed (Shatzman,
* Author for correspondence:
Tel: 91-11-26089688; Fax: 91-11-26088874
E-mail: sk608@rediffmail.com
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INDIAN J BIOTECHNOL,
OCTOBER 2003
phosphate dehydrogenase),
URA3 (orotidine 5'phosphate
decarboxylase),
TRP5
(tryptophan
synthetase),
LEU2
(p
isopropyl
malate
dehydrogenase), ARG2 (arginosuccinate lyase) and
CANI (canavanine). CANI confers sensitivity to
canavanine, an arginine analog that gets incorporated
in proteins, and is lethal to cells. It is an arginine
permease that allows canavanine to enter into host
cells. The CANI containing vectors can be used in
plasmid shuffling experiments where one version of a
gene, usually the wild type (WT), is replaced by
another version (the mutant) by selecting for the loss
of the WT vector on canavanine. This technique is
required if the original gene is an essential gene and
the cell cannot survive without it. A similar strategy is
followed with URA3 and a drug called 5-FOA
(fluroorotic acid) is used for this purpose. FOA is
converted into a toxin by URA3 and the presence of
URA3 on plasmid is lethal in the presence of FOA. In
this way, a URA3 plasmid can be cloned out of yeast.
FOA is very expensive and is used on very small
plates (Boeke et al, 1984). Following general category
of the yeast vectors are employed for different
purposes:
Integrative vectors. Yeast integrative plasmids
(YIps) consist of bacterial vector components and a
yeast gene with selectable marker. These cannot
survive in yeast as free plasmid as they lack an origin
of replication and a centromere.
Site-specific
integration of plasmid into host genome is mediated
by homologous recombination between chromosomal
DNA and vector DNA (Grallert et al, 1993). These
plasmids are used to carry foreign genes into the yeast
genome so that the selection pressure does not have to
be maintained and the gene will be expressed as a
single copy gene. Two different strategies have been
developed to integrate exogenous DNA into the yeast
chromosomes. The first approach involves the use of
YIps that lack an origin for autonomous replication
but carry sequences, which allow their integration to
chromosomes at high frequency. These plasmids are
linearized by a single restriction cut within the
complementary
yeast gene on the vector for
integrative gene conversion. A number of integrating
vectors have been used successfully to express a
variety of heterologous proteins in yeast. YIps with
two different yeast DNA sequences, one coding for
functional URA3 locus and the other coding for a
mutated "his3" gene, which is to be integrated into its
normal chromosomal site, have been constructed.
These contain two regions homologous to yeast
479
8gl11
Eco
Hind III
RI (4361)
Barn HI (375)
Sal (651)
1 Eco RI
2 Poll, dATP, dTIP/I
3 Ligase
(J
8gl11 Hind III
Replicating
vectors. Yeast replicating vectors
(YRps) contain prokaryotic plasmid DNA sequences
with part of a yeast DNA derived from YIp vectors
and also include chromosomal origin of replication.
The transformation efficiency of these plasmids is
2-3 fold higher than the YIp vectors. This high
frequency is thought to be due to presence of origin of
replication, which allows these vectors to replicate
autonomously. Such vectors are often referred as ARS
(autonomous
replicating
sequences)
vectors
(Stinchomb et al, 1979; Wohlgemuth & Bulboca,
1994). A prototype vector, YRp7 (Fig. 1), is 5.7 kb in
length (Stinchomb et al, 1979; Hitzeman et al, 1981).
However, often a rapid loss of these plasmids is seen
in growing cell population. Providing centromeric
(CEN) DNA sequences to YRp plasmids can solve
this problem. These sequences contain a central AT
rich region of 90 bp flanked by highly conserved
region of 11 and 14 bp respectively (Hieter et al,
1985; Caddle & Calos, 1994; Murkami et al, 1996).
Episomal vectors. Yeast episomal vectors (YEps)
contain prokaryotic sequences, a selectable yeast
marker gene and the entire 2 ~ plasmid also known as
"scp" plasmid. The 2 ~ plasmid is found in almost all
strains of S. cerevisiae in 50-100 copies per haploid
cell amounting to 2-3% of total chromosomal DNA
(Grimm et al, 1988; Smerdon et al, 1998). The most
striking structural feature of 2 ~ plasmid is the
presence of two inverted repeats of 599 bp which
divide the plasmid molecule into two non-identical
region. A recombination at these repeats yields two
forms (A and B) of this plasmid (Fig. 2). The 2 ~
plasmid possesses a single origin of replication and
replicates autonomously (Harteley & Donelson, 1980;
Smerdon et al, 1998).
2 Isolation of large
fragment
8gl11 Hind III
Barn HI (375)
Sal (651)
Fig. I-Construction
Eco RI
(2407)
Hpa I
Form A
(2964)
= 3912
bp
EcoRI
(2407)
Hpa I
(2964)
Form B
Eco RI A
Eco RI B
= 2407
= 4312
- 341 + 2006
- 2407 + 341
= 4072
= 2246
bp
bp
Fig. 2-Forms
A and B of the 2~ plasmid. The parallel lines
indicate the inverted repeats. Open bars represent the location of
the origin of replication. The locations of restriction sites have
been indicated, numbers represent the positions in form A
(Hartley & Donelson, 1980).
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INDIAN J BIOTECHNOL,
Artificial
chromosome.
Normal
eukaryotic
chromosomes are always linear. The plasmids, when
supplied with ARS, the CEN sequences and a
functional telomere, can get replicated in autonomous
manner and are stably maintained in linear form as an
extra artificial mini-chromosome (Murray & Szostak,
1986). While highly stable, these vectors are not
useful for expression of foreign proteins in yeast.
OCTOBER 2003
Protein
Reference( s)
481
pombe
Vector
Origin of replication
Promoter
Yeast marker
pART
pEVP II
pCHY21
pREP
pTL2M UpAL 7
pSL2M UpAL 7
pSLF
pSLF 101
pSM 112
ars I
2J..lplasmid ori
ars I
ars I
ars II stb
ars II stb
ars I
ars I
2J..lplasmid ori
adh promoter
adh promoter
fbp I promoter
nmt I promoter
hCMV promoter
hCMV promoter
CaMV promoter
CaMV-tet
SV 40 promoter
LEU 2
LEU 2
URA 3
LEU 2
neol LEU 21 URA 3
neo/ LEU 21 URA 3
LEU 2
LEU 2
LEU 2
pART IIN795
ars I
adh promoter
LEU 2
CaMV, cauliflower mosaic virus; nea, neomycin resistance; tet, tetracyclin resistance gene Giga-Hama & Kumagai, 1999
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INDIAN J BIOTECHNOL,
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SV40-T
stb
LEU2NRA3
Fig. 3-Structure
of the expression vectors. A. High-level
expression
vector pTL2M,
having hCMV promoter.
B.
Transducing vector pAL7/pAUS. HCMV-P, hCMV promoter,
SV40-P, SV40 promoter; SV40-T, SV 40 terminato; neo,
neomycin resistance gene; UTR, untranslated region; MCS,
multiple-cloning site; amp, ampicillin resistance gene; ori, origin
of replication; LEU21URA3, selection marker (Giga-Hama, 1997).
483
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INDIAN J BIOTECHNOL,
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The ability of yeast to perform various posttranslational modifications and targeting of proteins to
specific cell membranes has led to its evaluation as an
expression system for membrane proteins (Evans et
al, 1995; Grisshammer & Tate, 1995). Membrane
proteins
require
complex
post-translational
modifications
including
interaction
with other
proteins, such as molecular chaperones, to attain their
final conformation and insertion into membranes. Due
to the poor understanding of the factors that are
important for membrane protein insertion and folding,
there are still only a few examples of highly expressed
heterologous membrane proteins in yeast. Often, the
initial expression experiments are performed in a host
that is homologous to the source of the membrane
protein. Plant and fungal membrane proteins are more
readily expressed in S. cerevisiae than the mammalian
membrane proteins (Villalba et al, 1992; Reutz &
Gros, 1994; Grisshammer & Tate, 1995). However,
there are some reports on expression of human
membrane proteins, including the erythroid anion
exchanger AEI (Sekler et al. 1995), the emopamilbinding protein (Hanner et al, 1995), the multiple
drug resistance related P-glycoprotein (Evans et al,
1995), the f3-adrenergic receptor (Grisshammer &
Tate, 1995), the receptors for dopamine (Mak et al,
1994), transferrin, retinoid X (Mak et al, 1994) and
estrogen (Arnold et al, 1996), and the Neurospora
crassa plasma membrane H+-ATPase (Mahanty &
Searborrugh, 1996) by yeast. By using yeast system,
sufficient amounts of functional protein suitable for
biochemical,
pharmacological
and
biophysical
analysis
can
be
obtained.
The
vertebrate
neuroendocrine peptide, cholecystokinin (CCK), is
subjected
to a number
of post-translational
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INDIAN J BIOTECHNOL,
486
Protein
Reference
Human lipocortin I
Human blood coagulation factor Xllla
B-Glucuronidase
HIV type-I protein R
Human Bradrenergic receptor
Human D2s dopamine receptor
Human P-glycoprotein
Human liver epoxide hydrolase
Cytochrome P450
Human gastric lipase
Human antithrombin III
Human interleukin-6
Human placental alkaline phosphatase
Giga-Harna & Kumagai, 1999
OCTOBER 2003
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489
490
491
492
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493