JOURNAL of BIOTECHNOLOGY RESEARCH in TROPICAL REGION, Vol. 1, Oct.
2008 (Special Edition) ISSN: 1979-9756
Construction of a CSF3-Synthetic Gene for Recombinant
Human G-CSF Expression in Yeast Using a TBIO
(Thermodynamically Balanced Inside-Out) Method
Asrul Muhammad Fuad1*, Dian Fitria Agustiyanti1, Yuliawati1, Citra Fidyani1,
Aminah1, Adi Santoso1
1
Bioprocess Engineering Laboratory, Bioprocess Division, Research Center for Biotechnology,
LIPI. Cibinong Science Center-Jalan Raya Bogor Km. 46, Cibinong 16911-Bogor
Abstract
Human Granulocyte-colony stimulating factor (hG-
CSF or G-CSF) is a cytokine, which has therapeutic
applications. It is a hematopoietic growth factor that
stimulates proliferation of granulocytic cells called
granulopoiesis. It is used to increase neutrophilic
granulocytes or neutrophils level in the body. Neu-
trophils compose the majority of white blood cell
(WBC) components and play very important roles in
human defense against infections. Neutropenia is
an abnormal condition where there is very low num-
ber of WBCs in human body due to various causes
such as drug side-effects, vitamin B12 defi-ciency,
cancer chemotheraphy, virus infections and bone
marrow cells abnormality. Nowdays, neutronnpenia
could be avoided by the administration of recombi-
nant hG-CSF preparation. G-CSF is a monomer
protein encoded by a single gene called CSF3.
There are two variants of G-CSF found in the body.
However, both variants (177aa and 174aa) show
similar bioactivity. Recombinant hG-CSF has been
used widely in combination with various anticancer
drugs to fight cancers. It was used to avoid neutro-
penia and complication during and after chemothe-
rapy. This research had a goal to produce recombi-
nant(s) hG-CSF and its analogs (muteins) with im-
proved characteristic(s) using synthetic gene(s) that
code for this protein. The recombinant protein will be
expressed in a defined yeast expression system. To
achieve this goal, we firstly had to construct a
CSF3-synthetic gene containing optimized codon for
expression in Pichia pastoris. At present, we suc-
cessfully constructed a version of CSF3-synthetic
gene using a PCR-based technique called the TBIO
(Thermodynamically Balanced Inside-Out) method.
*Correspondence to: Dr. Asrul Muhammad Fuad
Tel: +62 21 8754587; Fax: +62 21 8754588
e-mail: asrul.fuad@yahoo.co.id
Construction of a CSF3-Synthetic Gene for Recombinant Human G-CSF Expression in Yeast Using a TBIO
(Thermodynamically Balanced Inside-Out) Method
A DNA sequence of 558bp long has been con-
structed using 14 oligonucleotides with an average
length of 60 nucleotides. The synthetic gene se-
quence and oligonucleotides used had been de-
signed to contain yeast’s codon preferences with the
DNAWorks3.1 program. It was cloned in a commer-
cial cloning vector and is undergoing DNA sequence
analyses.
Keywords: recombinant hG-CSF, CSF3, neutrope-
nia, neutrophils, granulopoiesis, synthetic gene,
TBIO, Pichia pastoris.
INTRODUCTION
G-CSF (granulocyte-colony stimulating factor) is
a haematopoietic growth factor that works by en-
couraging the bone marrow to produce more white
blood cells. Growth factors are special proteins pro-
duced naturally in the body. They can also be made
as drugs. One of the main side effects of chemothe-
rapy drugs is the reduction in the number of white
blood cells. This makes our body less able to fight
infection. There is a risk that one could develop a
serious infection, which might have to be treated in
hospital. If the number of blood cells (blood count)
is low when the next dose of chemotherapy is due,
then the chemotherapy may have to be postponed or
the dose lowered.
In this situation, G-CSF can be given to stimulate
the bone marrow to produce new white cells more
quickly after chemotherapy. This can shorten the
period during which the patient is at risk of develop-
ing a serious infection. G-CSF is not needed with all
types of chemotherapy treatment, as the white blood
cell count can often recover on its own. G-CSF may
sometimes be used before high-dose chemotherapy
to make the bone marrow produce more stem cells.
These extra stem cells can then be collected and
given back to you after high-dose chemotherapy
treatment. The stem cells then go back into the bone
marrow and produce blood cells.
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JOURNAL of BIOTECHNOLOGY RESEARCH in TROPICAL REGION, Vol. 1, Oct. 2008 (Special Edition)
Human G-CSF was coded from a single gene
called CSF3. It was mapped on the chromosome 17.
There are two isoforms of mature protein G-CSF
mostly found in the body, which are 177 aa
(NP_000750) and 174 aa (NP_757373) in lengths.
Both isoforms show similar bioactivities. There are
three different types of G-CSF commercially availa-
ble; lenograstim (Granocyte®), filgrastim (Neupo-
gen®) and pegylated filgrastim (Neulasta®). These
drugs work in a similar way. The molecules of the
pegylated filgrastim have more glycosylation that
helps the drug to work longer.
Chemical synthesis of DNA sequences provides
a powerful tool for creating, modifying and studying
gene function; such as studying its structure and
expression in a given host cell. In the past, the most
direct method to construct a synthetic gene were to
mix overlapping preformed double stranded DNA
and ligate them each other enzymatically. However,
the yield of the full-length product declines sharply
with increasing number of DNA duplexes. The more
common method is to construct separate DNA seg-
ments of the gene from a smaller number of DNA
duplex, amplify each fragment by sub-cloning into a
plasmid vector then ligate the fragments to give the
full-length gene. Although, this method could effi-
ciently produce the intermediate product for each
step, the intermittent sub-cloning and bacterial am-
plification steps make the procedure very tedious
and time-consuming. The more recent method in-
volves assembling of several synthetic primers or
oligonucleotides to produce up to 500 bp DNA se-
quence, assembled in a single-tube annealing and
ligation reactions.
In this study, we applied a method for a synthet-
ic gene construction called the TBIO (Thermody-
namically Balanced Inside-Out) method. This me-
thod, originally reported by Gao et al. (2003), offers
a very efficient method for gene construction with-
out the use of restriction and ligation procedures.
This method is a PCR-based single-step DNA syn-
thesis uses both primers in sense and anti-sense
strands each for half of the gene length. The primer
elongation will run on both directions. Thus, TBIO
bidirectional elongation must be completed for a
given outside primer pair before the next round of
bidirectional elongation can take place. The method
was reported to be successfully used in constructing
some DNA sequences of up to 1712 bp in length.
The same method was used to generate longer DNA
sequences in a slightly modified method (Xiong et
al., 2004). The method was reported to give high-
fidelity and cost-effective PCR-based two-step
DNA synthesis for construction of long segments of
DNA. The long DNA sequence (2,382 bp) was dis-
sected into five DNA segments of around 500 bp in
Fuad, et.al.
ISSN: 1979-9756
length. Each of which was constructed with the
TBIO method.
The purpose of this study was to synthesize a
yeast-codon-optimized CSF3 gene which might be
expressed extracellularly from a methylotrophic
yeast Pichia pastoris. An mRNA variant of human
CSF3 gene (variant-2) producing the hG-CSF mole-
cule isoform-b, was altered to obtain a CSF3 syn-
thetic gene with optimized-codon preference for
protein expression in P. pastoris. In this research,
we constructed a DNA sequence or an ORF (Open
Reading Frame) of hG-CSF gene, called CSF3, with
the TBIO method. The method was slightly mod-
ified in which the PCR reactions was run sequential-
ly with a given primer pair from the middle of the
gene. The PCR product was then used as template
for sequence elongation with the next primer pair of
the outside part of the sequence being generated.
The amino acid sequence of the hG-CSF iso-
form-b (NP757373) was used as template for gener-
ation of the synthetic gene sequence. The signal
peptide (first 30 amino acid) was excluded from the
sequence, resulting in a protein sequence of 174 aa
in length.
MATERIALS AND METHODS
Materials: The oligonucleotide primers were syn-
thesized by Generay Biotech. The high fidelity Pfu
DNA polymerase and dNTPs mix were purchased
from Fermentas. Cloning plasmid InstanCloneTM
was purchased from Fermentas. Plasmid DNA gel
extraction kit was from RBC. The XhoI and SalI
restriction enzymes were from Fermentas. T4 DNA
ligase was from Fermentas.
CSF3 Open Reading Frame (ORF) and primer
design: The CSF3 Open Reading Frame (ORF) was
generated based on the protein sequence of the iso-
form-b of hG-CSF molecule having 174 amino acid
residues. The protein sequence was retrieved from
the gene database (GenBank accession no.
NP_757373) or the protein database (SwissProt ac-
cession no. P09919-2). The synthetic gene excludes
the first 30 aa native signal peptide. The DNA frag-
ment of the synthesized CSF3 ORF was 522 bp in
length. However, in the design of the CSF3 ORF,
two restriction sequences (XhoI and SalI) were add-
ed at both ends of the synthetic gene as well as a
linker peptide (KREAEA) at the 5‟ end. The result-
ing ORF sequence was 558 bp in length. The protein
sequence was submitted to a software program,
DNAWorks 3.1, by which the oligonucleotide se-
quence (ORF) of the synthetic gene was then gener-
ated. The gene was optimized to contain P. pastoris
codon preference. Some parameters was set up for
2
JOURNAL of BIOTECHNOLOGY RESEARCH in TROPICAL REGION, Vol. 1, Oct. 2008 (Special Edition)
the required oligonucleotide synthesis, including
primer length (set at 60 nt), annealing temperature
(set at 60oC), codon frequency threshold (set at
10%) and the output mode was set to TBIO method
(thermodynamically balanced inside-out). The pro-
gram sent a detail output of the DNA sequence as
well as primer sequences required for construction
of the synthetic gene. There were 14 primers or oli-
gonucleotides which should be synthesized, each
having 60 nt (nucleotides) in average and an overlap
region varied from 18 to 25 nt between adjacent
primers.
CSF3 Open Reading Frame construction and ex-
perimental design: The synthetic gene was con-
structed according to a modified TBIO method (Gao
et al., 2003). The PCR-based primer extention me-
thod of the synthetic gene, which is the basic prin-
ciple of the TBIO method, was started from the
middle of the gene sequence. As shown in Fig. 1,
the 3‟-terminal ends of the first pair of 60 mer
sense- and antisense-strand TBIO primers (P7 and
P8) overlap in the middle of the synthetic gene se-
quence. The gene synthesis started at this point by
primer extension process. The PCR reaction was
continued with the next 60 mer pair of outer primers
(P6 and P9), both from sense- and antisense-strands.
The reaction was repeated with the next pair of pri-
mers. Those pairs of primers were added sequential-
ly to extend the sequence polymerization in both
directions of sense and antisense strands. The PCR
mix reaction uses 40 nM of each primer pairs, 0.2
mM dNTPs, 1x Pfu buffer and 1.25 U of Pfu DNA
polymerase in a 50 µl PCR mix reaction volume.
For the subsequent PCR reaction, 2.5 µl of the inner
DNA sequence was added (as template) into the
next PCR reaction with the next pair of primer for
DNA sequence elongation. The PCR cycles used
was 2 min for first denaturation at 95oC, for 1 min
of denaturation at 95oC, for 30 sec of annealing at
59oC, for 1 min of elongation at 72oC and 5 min for
final elongation at 72oC. The PCR reaction was set
for 25 cycles.
Cloning and analysis of the synthetic gene: The
PCR products of each sequential PCR reactions
were analyzed in a 1.5% agarose gel electrophoresis.
The final length of the synthetic DNA sequence be-
ing constructed is 558 bp. Into this PCR product, an
amount of 1 to 2 U of Taq DNA polymerase (Fer-
mentas) was added and the mixture was incubated at
72oC for at least 30 min. This process was carried
out in order to add an additional “A” (adenine) at
the 3‟-end of each double stranded synthetic DNA
sequence which has been produced. The DNA prod-
uct was then sub-cloned into a commercial A/T
cloning plasmid kit such as InstanCloneTM
Construction of a CSF3-Synthetic Gene for Recombinant Human G-CSF Expression in Yeast Using a TBIO
(Thermodynamically Balanced Inside-Out) Method
ISSN: 1979-9756
pTZ57R/T (Fermentas). The recombinant plasmid
was transformed into E. coli XL1-Blue. The recom-
binant clones were then selected on selection LB-
agar medium containing IPTG and X-gal. Positive
clones (white colonies) were selected, cultured in an
appropriate media and the recombinant plasmids
were extracted and analyzed. Enzymatic restriction
analysis was then carried out to analyze the recom-
binant plasmids using single and double-digestion
analysis (with XhoI and SalI). Cloning and trans-
formation into the cloning plasmid kit were carried
out according to protocols given by the producer.
Plasmid preparation and restriction analysis were
done using the general protocols for molecular clon-
ing according to Ausubel et al. (2002). The recom-
binant plasmid(s) harboring the correct DNA insert
was then submitted for DNA sequence analysis us-
ing appropriate primers.
RESULTS AND DISCUSSION
CSF3 Open Reading Frame (ORF) and primer
design: Human G-CSF is encoded by a single gene
called CSF3 belongs to IL-6 superfamily. The gene
is located in chromosome-17 and mapped at locus
17q11.2-q21 by in situ hybridization (Tweardy et
al., 1987). The gene produces 3 variants of mRNA
that resulted in 3 different types of preprotein hG-
CSF. However, there are two isoforms of mature
hG-CSF mostly found in the body; isoform-a (177
aa, GenBank accession no. NP-000750) and iso-
form-b (174 aa, GenBank accession no. NP-
757373). Although isoform-b lacks three amino ac-
ids VSE at position 66-68, both isoforms show simi-
lar bioactivity.
In this study, the protein sequence of the short ver-
sion of hG-CSF (isoform-b) was used as template to
generate the synthetic CSF3 gene sequence
(CSF3syn). The human CSF3 gene that codes for
hG-CSF was codon-optimized for expression in
yeast. However, the first 30 aa native peptide signal
was excluded in the synthetic gene design. Instead, a
linker peptide KREAEA was added at the N-
terminal of the sequence. The linker peptide
presents proteolytic cleavage site(s) that will be use-
ful for secretion of the recombinant protein in yeast
P. pastoris since the protein target will be fused
with a yeast-derived signal sequence factor- . Table
1 shows the hG-CSF protein sequence (isoform-b)
with the peptide linker. The sequence was used as
input and submitted into the DNA Works 3.1 pro-
gram (online) to generate the DNA sequence
needed. The synthetic gene was designed to contain
optimized codon preferences for expression in yeast
P. pastoris. The ORF of the synthetic gene generat-
ed by the program is shown in Table 2.
3
JOURNAL of BIOTECHNOLOGY RESEARCH in TROPICAL REGION, Vol. 1, Oct. 2008 (Special Edition) ISSN: 1979-9756

Two restriction sites, XhoI and SalI, were added at poly-His Tag at the C-terminal. However, the stop
both ends of the sequence for cloning purpose in the codon will be included for the construction of other
yeast expression vector. The stop codon was ex- version of gene without Tag. The resulting ORF
cluded since the sequence would be fused with a sequence was 558 bp in length.

Table 1. Polypeptide sequence of CSF3 (or hG-CSF) used as input for the synthetic gene
design using the DNA Works 3.1 program.

Polipeptide sequence
1 KREAEATPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELVLLGHSL
61 GIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFA
121 TTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP
181

Note: The KREAEA sequence is a peptide linker between signal sequence Factor-α and
the synthetic gene

Table 2. DNA sequence of the CSF3 synthetic gene as output from DNAWorks 3.1.

DNA sequence
1 AAGAGAGAGGCTGAAGCTACTCCACTAGGCCCAGCTTCTTCTTTGCCACAATCTTTTCTT
61 TTGAAGTGTTTGGAACAAGTTAGAAAGATTCAGGGTGATGGTGCTGCCTTGCAGGAAAAG
121 TTGTGTGCTACTTACAAGCTGTGTCATCCAGAAGAATTGGTCTTGCTGGGACATTCTTTG
181 GGTATTCCATGGGCTCCATTGTCTTCTTGTCCATCTCAAGCTCTGCAATTGGCTGGTTGT
241 TTGTCTCAGTTGCATTCTGGTTTGTTTCTGTACCAAGGATTGTTGCAAGCTTTGGAAGGT
301 ATTTCTCCAGAGTTGGGACCAACTTTGGATACTTTGCAACTTGATGTTGCTGATTTTGCT
361 ACTACTATTTGGCAACAAATGGAAGAACTAGGTATGGCTCCTGCTTTGCAGCCAACTCAA
421 GGTGCTATGCCAGCCTTTGCATCAGCTTTTCAGAGAAGAGCTGGTGGTGTTTTGGTTGCT
481 TCTCATTTGCAGTCTTTCCTAGAAGTTTCTTACAGAGTTTTGAGACATTTGGCTCAACCA
541

CSF3 Open Reading Frame construction with
TBIO method: The DNA Works 3.1 program also
generated 14 oligonucleotides or primers for the
gene construction in addition to generating the syn-
thetic gene sequence. Those primers have an aver-
age length of 60 nt (nucleotides). As seen in Fig. 1,
half of them have the sense-strand sequences and
the other half have the antisense-strand sequences
(Primer sequences not shown). Between the adja-
cent primers, there are overlap regions between 18
and 25 nt in length. However, the overlap regions
have been optimized to have equal Tm value (an-
nealing temperature), which is 60±1oC.
The TBIO-designed primer set was used for the
gene synthesis by a PCR-based method. The gene
was synthesized by seven-step sequential „inside-
out‟ bidirectional elongation reactions from the
middle to both ends, which are the N- and C-termini
of the synthetic gene sequence. A pair of TBIO pri-
mers was used in each elongation. This method effi-
ciently produced the desired DNA product, which
was the ORF of the synthetic gene as shown in Fig.
2.

Fuad, et.al. 4
JOURNAL of BIOTECHNOLOGY RESEARCH in TROPICAL REGION, Vol. 1, Oct. 2008 (Special Edition) ISSN: 1979-9756

p1
p2
p3
p4
p5
p6
p7

p8
p9
p10
p11
p12
p13
p14

Forward primer
Reverse primer

Fig. 1. Construction method of CSF3 synthetic gene using the TBIO (thermodynamical-
ly balanced inside-out) method. Primer extension was started from the middle part of
the gene, followed by exterior primer pairs. The process was carried out by sequential
PCR with each pair of primers. Half of the primers have the sense strand sequence,
whereas the other half have the anti-sense strand sequence.

(bp) 1 2 3 4 5 6 7 8 range. The program used to generate the synthetic

1000
600
500
400
300
200
100
Fig. 2. DNA Analysis of PCR products for the CSF3 syn-
thetic gene construction using the TBIO method. DNA
ladder 100pb (lane-1) and PCR products (lane-2 to 8):
P7~P8 (lane-2); P6~P9 (lane-3); P5~P10 (lane-4);
P4~P11 (lane-5); P3~P12 (lane-6); P2~P13 (lane-7);
P1~P14 (lane-8). The synthetic gene final product was
558 bp in length (lane-8).
The TBIO-designed primer characteristics were
shown in Table 3 (A-D), including frequency range,
Tm range, overlap length range and primer length
gene sequence, the DNA Works 3.1, has a range of
parameters that could be fixed or customized. For
example, when the primer length was set at a fix
value for 60 nt and the Tm range varies from 60 to
65oC, then the program sorted out a number 6 dif-
ferent sequences appropriate for each Tm value.
Then one sequence that showed the best Tm range
of 1.3oC was chosen, which was the sequence with
the Tm setting at 60oC. This means that the gap or
difference of annealing temperatures of each adja-
cent primers were not greater than that value
(60±1.3oC). The less the Tm ranges the better elon-
gation process in the PCR reactions.
The PCR product showed that the sequential
PCR reactions carried out according to this method
worked well (Fig. 2). However, when all of primers
were used in a single PCR mixture and was run in a
single-step PCR, no product was resulted (data not
shown). The primer length of 60 nt was chosen for
effective result and cost considerations. Xiong et al.
(2004) has successfully constructed a quiet long
gene sequence called vip3aI (2,382 bp). He reported
the effective cost that should be spent with 60 nt
primer was better than 90 nt primer for the vip3aI
gene synthesis. The cost was reduced to one third
using 60 nt primers compared to 90 nt primers.

Construction of a CSF3-Synthetic Gene for Recombinant Human G-CSF Expression in Yeast Using a TBIO
(Thermodynamically Balanced Inside-Out) Method 5
JOURNAL of BIOTECHNOLOGY RESEARCH in TROPICAL REGION, Vol. 1, Oct. 2008 (Special Edition) ISSN: 1979-9756

Table 3. Some characteristics of the oligonucleotides used for the synthesis of the CSF3 synthetic gene. (A) Codon fre-
quency range; (B) Annealing temperature range (Tm); (C) Overlap region range; (D) Oligos length.

A B C D
Frequency No. of Tm range No. of Overlap No. of oligos Length range No. of
range (%) codons (oC) overlaps length range oligos
(nt)
0-4 0 < 58 0 < 17 0 < 49 2
5-9 0 58 0 17 0 49-50 0
10-14 4 59 5 18 3 51-52 0
15-19 7 60 8 19 2 53-54 0
20-24 2 61 0 20 3 55-56 0
25-29 15 62 0 21 0 57-58 0
30-34 27 63 0 22 3 59-60 12
35-39 14 64 0 23 1 CSF3 61-62 0
40-44 30 65 0 24 0 63-64 0
45-49 25 66 0 25 1 65-66 0
50 56 26 0 67-68 0
27 0 69 0

Cloning and analysis of the synthetic gene: The
PCR-based gene synthesis with TBIO method re-
sulted in a final product of the target gene having
558 bp in length. Each step of the sequential PCR
reactions produced a set of DNA products with in-
cremental length, ranging from around 100 to 558
bp of the final product (Fig. 2). During all amplifi-
cation processes, Pfu DNA polymerase was used to
ensure the accuracy of the amplified DNA sequence
from its primers. However, the Pfu DNA polyme-
rase would produce a „blunt end‟ product only. In
view of cloning the synthetic gene product into an
„A/T‟ cloning vector, to the DNA product „A‟ over-
hangs were added at both ends by incubating the
synthetic gene product with Taq DNA polymerase
(at 72oC, for 1 h) prior to ligation process into the
cloning plasmid (pTZ57R/T).
Transformation of the ligation product has success-
fully produced E. coli (strain XL-1 Blue) transfor-
mants which harbor the recombinant plasmid con-
taining the putative synthetic gene sequence. Re-
striction analysis (with XhoI and SalI) was done to
some positive clones obtained and some of them
have shown the correct DNA insert (Figs. 3 and 4).

1 2 3 4 5 6 7

1 2 3 4 5 6 7 8 9 10 11 12 13

3000
2000
1500
1000 CSF3syn
750
500
250

Fig. 3. Recombinant plasmid miniprep which contains Fig. 4. Restriction analysis of recombinant plasmid
the CSF3 synthetic gene. Lane-1: plasmid w/o DNA in- TZ57R-CSF3syn with XhoI and SalI restriction enzymes.
sert (Control); lane 2-12: different clones of recombinant
plasmids.

Fuad, et.al. 6
JOURNAL of BIOTECHNOLOGY RESEARCH in TROPICAL REGION, Vol. 1, Oct. 2008 (Special Edition)
In conclusion, the DNA Works 3.1 program has
enabled everyone to design any DNA sequence or
gene of interest in a flexible manner by combining
methods (TBIO or TBC) and sequence optimization
for a synthetic gene construction. Availability of a
codon usage database referred by this program
might enhance the quality of the DNA sequence
output resulted from this program.
However, regardless of whether codon-optimized
sequences improve the yield of the protein of inter-
est, the TBIO method of PCR-based gene synthesis
provides a forceful alternative approach for engi-
neering DNA sequences for many other uses such as
construction of : (1) predicted genes/ cDNA that are
difficult to clone or the corresponding mRNA
sources are difficult to obtain; (2) alternatively
spliced gene variants; (3) newly designed prokaryo-
tic plasmids that can be used to create new strain of
microbes and (4) newly designed eukaryotic vectors
that can be used for transgenic studies, gene therapy
and DNA vaccines.
DNA sequencing of the CSF3 synthetic gene
(CSF3syn) for the sequence analysis is still in
progress. However, restriction analysis has shown
that the target gene having a correct length of
around 558 bp has been successfully synthesized.
The chance of obtaining a fully correct gene se-
quence seems to be high, since the high fidelity Pfu
DNA polymerase is used during the gene construc-
tion. Although there is also possibility to have some
mutated sequences, usually point mutation such as
deletion or substitution, it is less expected.
Construction of a CSF3-Synthetic Gene for Recombinant Human G-CSF Expression in Yeast Using a TBIO
(Thermodynamically Balanced Inside-Out) Method
ISSN: 1979-9756
ACKNOWLEDGEMENT
This research is supported by The Indonesian In-
stitute of Sciences (LIPI)‟s Competitive Research
Program 2008, sub-program the Post Genomic Mo-
lecular Farming.
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