WO2011151721A1

WO2011151721A1 - Process for production of fusion proteins using truncated e. coli thioredoxin

Info

Publication number: WO2011151721A1
Application number: PCT/IB2011/001246
Authority: WO
Inventors: Shardul Salunkhe; Bhaskarjyoti Prasad; Sriram Padmanabhan; Ketaki Sabnis Prasad; Veena Pai Raiker
Original assignee: Lupin Limited
Priority date: 2010-06-04
Filing date: 2011-06-03
Publication date: 2011-12-08

Abstract

The present invention relates in general to a process of production of recombinant heterologous proteins using fusion proteins of truncated E. coli thioredoxin. Further the invention relates to the fusion proteins of truncated thioredoxin to increase the production, activity, stability or solubility of recombinant heterologous proteins. The peptide or protein may be fused to the carboxyl terminus of the truncated thioredoxin molecule. The protein of interest includes PTH (1-34).

Description

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. In 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: 1 DNA 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 He He 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 He Ala Asp Glu Tyr Gin Gly Lys Leu Thr Val Ala Lys Leu Asn He 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, granulocyte- macrophage 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 N- terminus 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, beta- galactosidase, 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, TOP 10, 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 (l gm 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 pET- TTrx75

Example 4: Amplification and cloning of Parathyroid hormone (1-34) gene in pET- TTrx 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 min 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(₆o_0nm) 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 μg/ml) and Truncated Thioredoxin (TTrx; 100 μg/ml) were incubated with l OuM 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.

1 1. 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.