PROCESS FOR PRODUCTION OF FUSION PROTEINS USING TRUNCATED
E. COLI THIOREDOXIN
Field of the invention
The present invention pertains in general to a process of production of recombinant
heterologous proteins using fusion proteins of truncated E. coli thioredoxin protein. Further
the invention relates to the fusion proteins of truncated thioredoxin to increase the
production, activity, stability or solubility of recombinant heterologous proteins.
Background of the invention
With the advent of recombinant DNA technology, several proteins, peptides can be prepared
in prokaryotic or eukaryotic host cell using different expression systems. Among the
expression host system, bacteria like E. coli is the most popular system because it is easy to
manipulate E. coli. However, when bacteria are used as host cells for heterologous gene
expression, it leads to a few limitations, and several problems for example inefficient
translation of mRNA, instability of mRNA in E. coli, toxic effect of the protein being
expressed Due to their small size, peptides are unable to adopt stable and soluble
confirmations, and are subject to intracellular degradation by proteases present in the host
cell.
Several techniques are available to solve these problems. n one of the strategy, gene fusion
method is used to express the protein of interest. With this technique large amount of
heterologous protein is produced by fusing the protein of interest to the carboxy terminal end
of fusion partners for example, GST protein, FLAG peptide, a hexa-his peptide or
thioredoxin. U.S. Pat. No. 7,585,943 discloses fusion proteins for the expression and for
separation of the protein of interest using a peptide linker. Thioredoxin and thioredoxin like
molecules as fusion protein partner are disclosed in U.S. Pat. Nos. 5,292,646; 5,270,181;
5,646,016; 6,143,524; 5,760,189; 7,253,144.
PCT applications WO20071 12676 and WO20071 12677 describes a method of preparing
human parathyroid hormone 1-34 using full length thioredoxin as fusion protein tag. U.S.
Pat. No. 7,223,566 disclose thioredoxin and its truncated derivatives for the production of
polypeptide as inclusion bodies in bacterial host cells.
Proteins found or expressed as insoluble or inclusion bodies renders them biochemically
inactive, denatured or functionally and structurally compromised polypeptide. These
inclusion bodies require further processing in order to solubilize and refold the heterologous
protein. If these additional processes are not successful, then it leads to a very little or no
protein retaining the bioactivity, moreover these additional procedures are expensive and
technically difficult. Several methods are developed for the selective isolation of desired
protein. The purification of proteins produced by recombinant technology is often a serious
challenge and there is a continuing requirement for new and easier methods to produce
homogeneous preparations of recombinant proteins. Therefore there remains a need for an
improved method for production and purification of stable and soluble proteins.
Summary of the invention
In an aspect the invention relates to a process for the production of the heterologous protein
in E. coli the process comprises of:
a) preparing a fusion DNA comprising a first DNA fragment encoding a E. coli truncated
thioredoxin (TTrx) and a second DNA fragment fused in the frame encoding the
heterologous protein of interest,
b) cloning of the vector comprising the fusion DNA of step a,
c) expressing the fusion protein in E. coli cells in soluble form,
d) obtaining protein from the fusion protein and
e) purifying the protein.
In another aspect, the invention is related to a fusion protein DNA sequence comprising an E.
coli truncated thioredoxin (TTrx) protein sequence fused to a DNA of the heterologous
protein of interest.
In another aspect, the invention provides a process for the production of heterologous protein
using a truncated thioredoxin of 75 amino acids as a fusion tag.
Description of the drawings
Figure 1: Amplification of TTrx gene
Figure 2: Clone map of pET- Truncated Trx - gene of interest fusion plasmid
Figure 3: Thioredoxin Purification
Figure 4 : Truncated thioredoxin (TTrx) purification
Figure 5 : Assay for oxidative activity of thioredoxin and truncated thioredoxin(TTrx)
purified proteins
DESCRIPTION OF SEQUENCE ID
SEQ ID NO: 1DNA sequence of E.coli Thioredoxin
atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg
gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc ccgattctgg atgaaatcgc
tgacgaatat cagggcaaac tgaccgttgc aaaactgaac atcgatcaaa accctggcac tgcgccgaaa tatggcatcc
gtggtatccc gactctgctg ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg
aaagagttcc tcgacgctaa cctggcc
SEQ ID NO: 2 DNA sequence of truncated E.coli Thioredoxin - Trx75
atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg gacggggcga tcctcgtcga
tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc ccgattctgg atgaaatcgc tgacgaatat cagggcaaac
tgaccgttgc aaaactgaac atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggt
SEQ ID NO: 3 Amino acid sequence of Trx75
Met Ser Asp Lys e e His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp
Gly Ala He Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met He Ala Pro He Leu
Asp Glu e Ala Asp Glu Tyr Gin Gly Lys Leu Thr Val Ala Lys Leu Asn e Asp Gin Asn
Pro Gly Thr Ala Pro Lys Tyr Gly He Arg Gly
SEQ ID NO: 4 DNA sequence of PTH (1-34)
tctgtgtccg agattcagtt aatgcataac cttggcaaac atttgaactc catggagcgt gtagaatggc tgcgtaagaa
gttgcaggat gtgcacaatt tttaa
SEQ ID NO: 5 Amino acid sequence of PTH (1-34).
Ser Val Ser Glu He Gin Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val
Glu Trp Leu Arg Lys Lys Leu Gin Asp Val His Asn Phe
Detailed description of the invention
The process of the invention provides the production of heterologous peptides or proteins in a
soluble and stable form in E. coli cells.
Specifically, the invention provides a method for producing a polypeptide in the soluble form
comprising:
a) obtaining a fusion DNA comprising a first DNA encoding a truncated thioredoxin (TTrx)
and a second DNA fused in the frame encoding the heterologous protein of interest,
b) cloning of the vector comprising the fusion DNA of step a,
c) expressing the fusion protein in E. coli cells in soluble form,
d) obtaining protein of interest from the fusion protein and
e) purifying the protein of interest.
The truncated thioredoxin (TTrx) of the invention refers to C terminal truncated E. coli
thioredoxin protein. The TTrx includes but not limited to 74(TTrx-74), 75(TTrx-75),
80(TTrx-80) amino acids from the N terminal of the full length TTrx. The truncated TTrx
retains the property of thermostability of fusion proteins. The fusion proteins obtained using
truncated thioredoxin (TTrx) are obtained as soluble protein. The fusion protein of the TTrx
has the properties of the thioredoxin like protein of higher expression, solubility and
thermostability of the fusion constructs.
Due to small molecular weight of the truncated thioredoxin protein of ~8 kDa, the molar ratio
with respect to the protein of interest will result in giving greater yield of protein of interest.
For example in the case of PTH (1-34). The truncated thioredoxin protein does not have
redox activity. One of the major disadvantages of full length thioredoxin protein is that it
causes oxidation of proteins. Therefore, by using truncated thioredoxin, the protein of interest
would not get oxidized thus giving rise to improved yield of protein of interest particularly
applicable to peptides and proteins that are amenable to oxidation.
According to the present invention, the DNA sequence encoding a heterologous peptide or
protein selected for expression in a recombinant system is fused to a truncated thioredoxin
DNA sequence for expression in the host cell. A truncated thioredoxin DNA sequence is
defined herein as a DNA sequence encoding a protein or fragment of a protein characterized
by an amino acid sequence having 1-75 amino acids having nucleotide sequence as shown in
SEQ ID NO:2.
The invention produces fusion proteins which retain the desirable characteristics of a
truncated thioredoxin protein i.e. thermostability, solubility, a high level of expression and
lack of oxido-reductase property.
The invention is not limited to any specific type of peptide or protein. A wide variety of
heterologous genes or gene fragments may be used in forming the fusion sequences of the
present invention. For example, hormones, cytokines, growth or inhibitory factors, enzymes,
modified or wholly synthetic proteins or peptides can be produced according to this
invention.
The term "protein of interest" as used herein refers to any protein or peptide the production
of which is desirable. In one embodiment, the protein or peptide is biologically active.
Examples of proteins of interest include, but are not limited to, interleukins i.e. IL-1 1, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, IL-15, IL-18, reteplase, parathyroid hormone,
thrombopoietin, epidermal growth factor, basic fibroblast growth factor, granulocytemacrophage
colony stimulating factor, granulocyte colony stimulating factor, macrophage
colony stimulating factor, platelet-derived growth factor, interferons including IFN-alpha,
IFN-beta, IFN-gamma, cleavage enzymes such as Factor Xa, thrombin, OmpT, TEV protease
and others.
In another embodiment, the protein of interest is human parathyroid hormone PTH (1-34)
and PTH (1-84).
The terms "fusion protein" as used herein, refers to polypeptides and proteins which
comprise a protein of interest, a fusion partner and a linker peptide with a cleavage site
interposed there in between. In one embodiment, the protein of interest is linked to the Nterminus
of the peptide linker and the fusion partner is linked to the C-terminus of the peptide
linker. In another embodiment, the protein of interest is linked to the C-terminus of the
peptide linker and the fusion partner is linked to the N-terminus of the peptide linker.
The term "fusion partner," as used herein, refers to any protein or peptide the inclusion of
which in a fusion protein is desirable.
In one embodiment the fusion partner is Truncated thioredoxin. The fusion partner imparts an
improved characteristic to the fusion protein, e.g., ease of purification, stability, solubility,
and the like.
In a further embodiment, a fusion protein may comprise more than one protein of interest
and/or more than one fusion partner, each separated by a peptide linker. In these
embodiments, the multiple proteins of interest may be the same or different, the multiple
fusion partners may be the same or different, and the multiple peptide linkers may be the
same or different.
The fusion partner may further include one or more affinity peptide which may ease
purification of the protein of interest. Examples of affinity peptides include, but are not
limited to, glutathione-S-transferase (GST), maltose binding protein (MBP), hexahistidine,
T7 peptide, ubiquitin, Flag peptide, c-myc peptide, polyarginine, polycysteine,
polyphenylalanine, BTag, galactose binding domain, cellulose binding domain (CBD),
thioredoxin, staphylococcal protein A, streptococcal protein G, calmodulin, betagalactosidase,
chloramphenicol acetyltransferase, S-peptide, streptavidin, His-tag, Strep-tag
and slyD.
A fusion sequence of a truncated thioredoxin sequence with a desired protein of interest
sequence according to this invention may optionally contain a linker peptide inserted
between the truncated thioredoxin sequence and the selected heterologous protein of interest.
The term "peptide linker," as used herein, refers to a specific amino acid sequence which
comprises an enzyme cleavage site which is recognized and cleaved by an enzyme. Cleavage
at the selected enzyme cleavage site enables separation of the heterologous protein of interest
from the truncated thioredoxin fusion protein to yield the mature heterologous protein of
interest. The mature protein may then be obtained in purified form, free from any polypeptide
fragment of the truncated thioredoxin protein to which it was previously linked. Any desired
cleavage site, of which many are known in the art, may be used for this purpose. For
example, the selected enzyme cleavage site may include sites for cleavage by a proteolytic
enzyme, such as enterokinase, Factor Xa, trypsin, collagenase, TEV protease and thrombin.
In one embodiment of this method, if the resulting fusion protein is cytoplasmic, the cell can
be lysed by conventional means to obtain the soluble fusion protein. More preferably in the
case of cytoplasmic fusion proteins, the method includes releasing the fusion protein from the
host cell by applying osmotic shock or freeze/thaw treatments to the cell. In this case the
fusion protein is selectively released from the interior of the cell via the zones of adhesion
that exist between the inner and outer membranes of E. coli. The fusion protein is then
purified by conventional means. As yet a further step in the above methods, the desired
protein can be cleaved from fusion with the truncated thioredoxin protein by conventional
means such as enterokinase digestion.
E. coli cells are transformed with suitable vector containing the fusion gene for the
production of fusion protein. Various strains of E. coli may be used for the process of the
present invention such as cells which are protease deficient strains such as BL21, ER2566
and the protease expressing strains of K12 derivatives such as HB101, JM109, LE392, C600,
TOP10, DH5 alpha and the like.
As yet another embodiment there is provided a method for increasing the expression of
soluble recombinant proteins. The method includes culturing the E.coli cells under suitable
conditions to produce the fusion protein. The fermentation may be carried out in fed-batch or
fed-mode under conditions to produce the truncated thioredoxin fusion proteins. Improved
expression of the proteins using the fusion DNA of the present invention depends on various
parameters of the fermentation process. Some of the parameters are fermentation media,
concentration of the inducer, nutrient feed rate.
Preferably the feed medium comprises carbon source and nitrogen source. The carbon source
may comprise glucose, glycerol, sorbitol, maltose, sucrose or starch, mannitol. The nitrogen
source may comprise ammonia, nitrate, peptone, soya peptone, yeast extract or tryptone. The
feed medium comprises of antibiotics and inorganic phosphates and trace elements.
The feed medium may comprise antibiotics such as ampicillin or tetracycline or any other
antibiotic such as kanamycin, tetracycline, chloramphenicol, hygromycin, carbenecillin and
the like depending on the antibiotic marker gene embedded in the vector.
The fusion protein accumulated in the cytoplasm of the cells may be released by
conventional bacterial cell lysis techniques and purified by conventional procedures
including selective precipitations, solubilizations and column chromatographic methods. The
truncated thioredoxin fusion proteins may be selectively released from the cell by osmotic
shock or freeze/thaw procedures. A simple centrifugation following this release removes the
majority of bacterial cell-derived contaminants from the fusion protein preparation. The
fusion protein may be further purified by well-known conventional methods.
The fusion protein is further treated with a proteolytic enzyme to release the yield the mature
heterologous peptide or protein. For example, the enzymatic cleavage may be done by a
proteolytic enzyme, such as enterokinase, Factor Xa, trypsin, collagenase, and thrombin.
In an embodiment of the invention the mature protein may then be obtained in purified form,
free from any polypeptide fragment of the truncated thioredoxin protein fragments to which it
was previously linked. The protein of interest may be purified using one or more purification
step. The purification techniques include affinity chromatography, metal affinity
chromatography, hydrophobic interaction chromatography, ion exchange chromatography,
Size exclusion chromatography and others. The sequence of the chromatography may be in
any order depending on the protein of interest and nature of impurities.
Other aspects and advantages of the present invention will be apparent upon consideration of
the following detailed description of preferred embodiments thereof.
Example 1. Thioredoxin purification (Figure 3)
Full length thioredoxin molecule was expressed from pET32a commercially available vector
from Novagen, USA. This protein has a his tag attached at its C terminus and hence was
purified using Nickel affinity purification in a single step upon expression in E. coli
BL21(DE3) cells after IPTG induction fro 4 hours at 37 deg C. the full length thioerdoxin
moelcule come sas a soluble protein in these E. coli induced cells and such a supernatant was
loaded on a 10 mM Tris. CI, pH 8.0 equilibrated Nickel agarose column. The bound proteins
were then eluted by using increasing concentration of imidazole. The thioredoxin protein was
eluted with 200 mM concentration of imidazole. The purified protein was analyzed on 13.5%
SDS PAGE followed by Coomassie staining or silver staining.
Example 2. Truncated thioredoxin (TTrx) purification (Figure 4)
TTrx was expressed in BL21(DE3) cells after IPTG induction for 4 hours at 37 Deg C from
the clone pET-TT. tTRX moiety was purified by two step purification process. tTRX
inclusion bodies were solubilized in 8M urea in lOOmM Tris.Cl pH 8.8 (lgm IB in 20 ml). It
was refolded in 10 mM Tris.Cl pH 8.0 by fast dilution and kept for refolding at slow stirring
for 16-18 hrs. It was diafiltered against 20 mM Tris.Cl pH 8.0 and loaded on Q sepharose FF.
Q-Sepharose column was equilibrated with 20mM Tris pH 8.0 and elution buffer was 20 mM
Tris.Cl, 1M NaCl pH 8.0. Elution was carried out in 0-100% B gradient in 70 CVs. Q elutes
between 30 to 50 mS/cm range were pooled and loaded on Phenyl FF (high sub) in 20 mM
Tris.Cl pH 8.0 containing 1M NaCl. Protein was eluted by 50-100% B gradient in 25 CVs.
TTrx was -95% pure in phenyl elutes as analyzed by SDS-PAGE.
Example 3 : Truncated Thioredoxin 75 (TTrx75) protein
Amplification and cloning of Truncated Thioredoxin (TTrx75): Truncated thioredoxin
(TTrx75) gene was amplified using pET32a gene as template and was cloned into pET21a
vector at Ndel/BamHl sites. Primers were designed in such a way that a truncated version of
Thioredoxin will get amplified and will contain 1 to 75 amino acids of thioredoxin molecule.
Primers for amplification:
FP for amplification of Truncated thioredoxin (TTrx75) gene
5' CCG CCG GAA TTC CAT ATG AGC GAT AAA ATT ATT CAC CTG ACT 3'
RP for amplification of Truncated thioredoxin (TTrx75) gene
5' CCG CCG GAA TTC GGA TCC ACC ACG GAT GCC ATA TTT CGG CGC AGT 3'
PCR conditions: The TTrx75 gene was PCR amplified using Taq DNA polymerase from
Bangalore Genei Pvt. Ltd (Bangalore, India) with the following amplification conditions.
Initial denaturation of 4 min at 94 °C followed by 5 cycles of 94 °C for 30 sec, 43 °C for 30
sec and 72 °C for 30 sec and 25 cycles with annealing temperature of 60 C for 30 sec. After
a final extension of 7 min at 72 C, the PCR amplified product was checked on 1% agarose
gel (Fig 1), purified and then digested with Ndel/EcoRI and ligated to the pET21a vector at
the similar sites.
The ligation mix was used to transform competent DH5 alpha cell line and the resultant 10
colonies were inoculated in 3 ml LB for overnight incubation in shaker for 16 h. Plasmid
DNA was isolated from the cultures and restriction analysis was done to confirm the release
of insert by Ndel/BamHI digestion (Fig 2). The resultant clones were designated as pETTTrx75
Example 4 : Amplification and cloning of Parathyroid hormone (1-34) gene in pETTTrx
vector:
PTH (1-34) gene was amplified using synthetic DNA as template and was cloned into
pET21a-TTrx75 vector at BamHI/Hindlll sites
Primers used for amplification of PTH gene:
FP for cloning of PTH (1-34) gene:
5' CCG CCG GGA TCC GAT GAT GAT GAT AAA TCT GTG TCC GAG ATT CAG TTA
3'
RP for cloning of PTH (1-34) gene:
5' CCG CCG GAA TTC AAG CTT TTA AAA ATT GTG CAC ATC CTG 3'
Primers were designed so that PTH (1-34) will be cloned as C terminal fusion to TTrx75
gene with Enterokinase cleavage site (DDDDK) and after enterokinase cleavage of TTrx75-
PTH (1-34) fusion protein, PTH (1-34) will get released with authentic N terminus.
PCR conditions:
The PTH (1-34) gene was PCR amplified using Taq DNA polymerase from Bangalore Genei
Pvt. Ltd (Bangalore, India) with the following amplification conditions. Initial denaturation
of 4 minutes at 94 °C followed by 5 cycles of 94 °C for 30 sec, 43 °C for 30 sec and 72 °C
for 30 sec and 25 cycles with annealing temperature of 60 C for 30 sec. After a final
extension of 7 in at 72 °C, the PCR amplified product was checked on 1% agarose gel,
purified and then digested with BamHI/Hindlll and ligated to the pET-TTrx75 vector at the
similar sites.
The clones were confirmed with restriction enzyme digestion and designated as pTTPTH.
Example 5 : Thermostability of the TTrx fusion protein:
The soluble fraction of sup was used for thermostability studies. The sample was incubated at
80 °C for 15 min. After incubation, the sample was centrifuged at 13000 rpm for 10 min. The
supernatant fraction was separated and was used for further analysis by Agilent bioanalyzer
2100. The result showed that even after heating of fusion protein at 80 °C, the protein did not
get precipitated (Fig 3 lane 2).
Enterokinase digestion:
The supernatant fraction of above experiment was used for digestion with enterokinase
(Novagen). The 500 of supernatant was incubated with 1.5 U of enterokinsae enzyme
with 10 mM Tris buffer and 1 mM Calcium Chloride. The reaction mix was incubated at 37
°C for lh. After digestion of fusion protein, the samples were loaded on an agilent 2100
bioanalyzer. PTH (1-34) was released after enterokinase digestion as seen Fig 3, lane 3.
Example 6 : Expression of fusion protein
TTrx75-PTH (1-34) fermentation was carried out in the fed batch mode using defined
medium comprised of salts like potassium phosphate and ammonium phosphate. Glucose,
mannitol, sorbitol or glycerol was used as the source of carbon and energy. Inoculum was
grown in two stages before it was inoculated in to the fermentation medium. Fermentation
process parameters followed were that of typical E. coli fermentation process i.e. pH ~ 6.5 to
7.5, temperature ~ 25 °C to 42 °C, aeration 0.5 to 2 vvm, etc. Nutrient feed medium was also
composed of potassium and ammonium phosphate salts and glucose, mannitol, sorbitol or
glycerol. Feed strategy adopted was "linear feed strategy". The feed strategy helped to keep
the acetate formation below 4 g/L during the entire batch. Inducer used was IPTG at the
concentration ranging from 0.05 mM to 2 mM. Fermentation batch time was ~ 12 to 14
hours, while cell density achieved was OD(6o0nm) HO. Growth inhibition was not observed in
spite of inducer addition, this helped to achieve higher biomass along with expression of
target protein. This resulted in higher yield of the process.
Example 7: Isolation of fusion protein
The fermentation broth was harvested and centrifuged at 7000 rpm at 4 °C for 10 minutes
and spent medium was collected separately. The induced cell pellet was processed for
disruption as detailed bellow.
The induced cell pellet was resuspended in 10 mM Tris.Cl, pH 8.0 at an OD(6oo) nearly 100
and then passed through the cell homogenizer at 800-900 (bars) for two passages such that
the turbidity of the solution after homogenization was reduced by 85 to 95 % which was
indicative of bacterial cell lysis. The entire process was carried out in cold by keeping cell
suspension as well as cell lysis on ice. The homogenizer was also connected to chilled water
line as an alternative.
The cell lysate was centrifuged at 10000 rpm for 20 minutes in cold and the soluble and the
insoluble proteins were separately collected. The soluble fraction contains the TTrx75-PTH
(1-34) and stored for further downstream process.
Example 8.: Assay for Oxidative activity of Thioredoxin and truncated thioredoxin
proteins : The method followed is as per Jung et al. Free radical biology and medicine Vol.
31(4), pp 479-89, (2001); Shankar et al. Radiation Research 160(4), 478-487 (2003) (Figure
5).
Thioredoxin (TRX; 100 g/ml) and Truncated Thioredoxin (TTrx; 100 g ml) were
incubated with lOuM 2', 7' - Dichlorofluorescin diacetate (DCFDA) at 37°C for lhr. To
increase the levels of oxidation, TRX and TTrx were preincubated with DCFDA at 80°C for
15mins and further kept for incubation at 37°C for lhr. On oxidation, the non-fluorescent
DCFDA gets converted to fluorescent DCF which is a measure of extent of oxidation. After
incubation, fluorescence was quantified using excitation at 488 nm and emission at 525 nm
respectively. Quantification of the levels of DCFDA fluorescence was assessed on a relative
scale of fluorescence units. Values represent means ± S.E. of DCFDA fluorescence from
triplicates.
Results: Incubation of TRX and TTrx caused oxidation of DCFDA as seen by the increase in
fluorescence when compared to the buffer control. However, in direct incubation group, TTrx
showed 15% reduced oxidation when compared to TRX. Heat treatment of TRX and TTrx
caused enhanced oxidation of DCFDA as compared to the direct incubation group.
Nonetheless, TTrx showed almost 75% reduced oxidation when compared to TRX.
CLAIMS
1. A process for the preparation of heterologous protein in E. coli comprising the steps of:
a) preparing a fusion DNA comprising a first DNA encoding a truncated thioredoxin and a
second DNA fused in the frame encoding the heterologous protein of interest,
b) cloning of the vector comprising the fusion DNA of step a,
c) expressing the fusion protein in E. coli cells in soluble form,
d) obtaining protein from the fusion protein and
e) purifying the protein.
2. The process as claimed in claim 1, wherein the truncated thioredoxin is 75 amino acid
protein having nucleotide sequence of SEQ ID 2.
3. The process as claimed in claim 1, wherein the heterologous protein of interest is selected
from the group comprising cytokines, growth stimulating factors, hormones, interferons,
interleukins, enzymes.
4. The process as claimed in claim 1, wherein the heterologous protein of interest is selected
from the group comprising parathyroid hormone (1-34), parathyroid hormone (1-84),
reteplase, interferon, IL-2, IL-3, IL-4, IL-5, IL-6, IL-1 1, GCSF.
5. The process as claimed in claim 1, wherein the heterologous protein of interest is
parathyroid hormone (1-34).
6. The process as claimed in claim 1, wherein the fusion DNA further comprises an
Enterokinase cleavage site.
7. The process as claimed in claim 1, wherein the protein is purified using one or more
chromatographic purification technique selected from the group consisting of affinity
chromatography, metal affinity chromatography, hydrophobic interaction chromatography,
ion exchange chromatography and size exclusion chromatography.
8. A fusion DNA comprising a first DNA encoding a truncated thioredoxin and a second
DNA fused in the frame encoding the heterologous protein of interest
9. The fusion DNA as claimed in claim 8, wherein the truncated thioredoxin is 75 amino acid
protein having nucleotide sequence of SEQ ID 2.
10. The fusion DNA as claimed in claim 8, wherein the truncated thioredoxin is 75 amino
long protein having amino acid sequence of SEQ ID 3.
11. The fusion DNA as claimed in claim 8, wherein the heterologous protein of interest is
selected from the group comprising cytokines, growth stimulating factors, hormones,
interferons, interleukins,
12. The fusion DNA as claimed in claim 8, wherein the heterologous protein of interest is
selected from the group comprising parathyroid hormone (1-34), parathyroid hormone (1-
84), interferon, IL-2, IL-3, IL-4, IL-5, IL-6, IL-1 1, and GCSF.