WO2013076657A1

WO2013076657A1 - Map fusion protein

Info

Publication number: WO2013076657A1
Application number: PCT/IB2012/056583
Authority: WO
Inventors: Shardul Salunkhe; Sriram Padmanabhan; Jitendra Kumar
Original assignee: Lupin Ltd
Current assignee: Lupin Ltd
Priority date: 2011-11-22
Filing date: 2012-11-21
Publication date: 2013-05-30
Anticipated expiration: 2014-05-22
Also published as: IN2014MN00935A

Abstract

The present invention relates in general to a process of production of recombinant heterologous proteins in E. coli expression system using E. coli, Yeast, fungi or mammalian methionine amino peptidase (MAP) protein as a fusion tag. Further the invention relates to the fusion proteins of MAP protein.

Description

MAP FUSION PROTEIN

FIELD OF THE INVENTION

The present invention relates to a process for producing heterologous proteins of interest using methionine aminopeptidase (MAP) protein as a fusion tag. Further the invention relates to fusion protein DNA sequences comprising MAP protein.

BACKGROUND OF THE INVENTION Recent advances in techniques related to recombinant DNA cloning in last two decades has led to increase in the number of proteins expressed in large scale. These proteins have therapeutic, diagnostic or industrial importance. The recombinant proteins can be expressed in variety of different expression systems like bacterial, fungal, yeast and mammalian cell systems. Each of the above systems has their own advantages and disadvantages. Though mammalian expression systems will express correctly folded proteins, the overall yields of protein of interest in this expression system are very low and the cost of production is relatively highest in comparison to other expression system.

The cloning and expression of foreign proteins in E. coli is highly efficient since E. coli offers high productivity, high growth and production rate, ease of use and economical. E. coli facilitates protein expression by its relative simplicity, is inexpensive, fast growth, well- known genetics and the large number of compatible tools available for biotechnology. However, there are a few disadvantages like lack of post-translational modifications, lack of proper secretion system for efficient release of produced protein into the growth medium, inefficient cleavage of amino terminal methionine which can result in lower protein stability and increased immunogenicity, limited ability to facilitate extensive disulphide bond formation, improper folding resulting in inclusion body formation etc. However, when heterologous proteins are expressed in E coli in high levels, recombinant proteins are frequently expressed in E. coli as insoluble protein aggregates termed as "inclusion bodies". In general small proteins (>30 kDa) that are simple monomeric proteins can be found in the soluble fractions of bacterial extracts. In contrast, proteins (<30 kDa) or proteins that have complex secondary or tertiary structures are typically insoluble and are predominantly found in inclusion bodies. Although initial purification of inclusion body material is relatively simple, the protein must be subsequently refolded into an active form which is typically a cumbersome process. If these additional procedures are not successful then little or no protein activity is recovered from the host cells. Thus, it is much more desirable to express the recombinant protein in soluble form.

Several approaches, including protein fusions, chaperone co-expression, and promoter alterations, have been used to overcome these problems (Zhang Y, Olsen DR, Nguyen KB, Olson PS, Rhodes ET, Mascarenhas D, 1998, Expression of eukaryotic proteins in soluble form in Escherichia coli, Protein Expr. Purif. 12, 159-165; Thomas JG, Baneyx F, 1997 Divergent effects of chaperone overexpression and ethanol supplementation on inclusion body formation in recombinant Escherichia coli, Protein Expr. Purif. 1 1 , 289- 296). Unfortunately, these methods are not widely applicable. One of the strategies to prevent formation of protein aggregates is to tag the protein of interest with a fusion protein with a protein known to have been soluble at very high levels. Several commercial fusion protein tags are available in market like Intein, maltose binding protein (Pryor KD, Leiting B1997 High-level expression of soluble protein in Escherichia coli using a 6X His-tag and maltose-binding-protein double-affinity fusion system, Protein Expr. Purif. 10, 309-319.), Glutathione S transferase (Nygren PA, Stahl S, Uhlen M 1994 Engineering proteins to facilitate bioprocessing, Trends Biotechnol. 12,184-188.), SUMO tags (Marblestone JG, Edavettal SC, Lim Y, Lim P, Zuo X, Butt TR 2006 Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO, Protein Sci. 15, 182-189.), ZZ tag, NusA (Marco VD, Stier G, Blandin S, Marco AD 2004 The solubility and stability of recombinant proteins are increased by their fusion to NusA, Biochem. Biophys. Res. Commun. 322, 766-771 .), Ubiquitin (Catanzariti AM, Soboleva TA, Jans DA, Board PG, Baker RT 2004 An efficient system for high-level expression and easy purification of authentic recombinant proteins, Protein Sci. 13, 1331-1339.) etc.

Methionine aminopeptidases are ubiquitously distributed in all living organisms. The removal of the N terminal methionine is a critical step for protein modifications that are important in controlling protein subcellular localization and/or protein degradation. Two distantly related MetAP enzymes, type 1 and type 2, are found in eukaryotes, while prokaryotes express only one type of MAP. MetAP I exit in eubacteria while MetAP II exists in archae ( Li X, Chang YH 1996, Evidence that human homologue of a rat initiation factor -2 associated protein (p67) is a methionine aminopeptidase Biochem Biophysics Res Comm 227-1 , 152-9 ; Dummitt B, Micka W, Chang YH 2003 N-terminal methionine removal and methionine metabolism is Saccharomyces cerevisiae J Cell Biochem 89:964- 974). N-terminal methionine removal in bacteria is a two step process requiring the removal of the N formyl group by polypeptide deformylase first followed by cleavage of the N terminal methionine when the adjacent amino acid is small. Both the above steps appear to be essential for bacterial cell viability. Failure to remove the N terminal methionine can lead to inactive enzymes (e.g. glutamine phosphoribosylpyrophosphate aminotransferase and N terminal nucleophile hydrolase).

The invention relates to use of MAP protein as a fusion tag to obtain a soluble protein of interest in E.coli cells.

SUMMARY OF THE INVENTION

In an aspect the invention relates to a process for the production of the heterologous protein in E. coli cells, the process comprises of:

a) preparing a fusion DNA comprising a first DNA fragment encoding MAP protein 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 MAP 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 as a soluble fusion protein using a MAP protein as a fusion tag.

DESCRIPTION OF THE DRAWINGS AND SEQ ID.

Figure 1 : Schematic representation of vector map of pET21 -MapF SEQ ID NO. 1 : DNA sequence of MAP protein

Sequence ID no. 1 : MAP DNA sequence (200230—200365), 795 bp

atggctatctcaatcaagaccccagaagatatcgaaaaaatgcgcgtcgctggccgactggctgccgaagtgctggagat gatcgaaccgtatgttaaaccgggcgtcagcaccggcgagctggatcgcatctgtaatgattacattgttaatgaacaac acgcggtttctgcctgcctcggctatcacggctatccgaaatccgtttgcatctctattaatgaagtggtgtgccacggt atcccggacgatgctaagctgctgaaagatggcgatatcgttaacattgatgtcaccgtaatcaaagatggtttccacgg cgatacctcgaaaatgtttatcgtcggtaagccgaccatcatgggcgaacgtctgtgccgcatcacgcaagaaagcctgt acctggcgctacgcatggtaaaaccaggcattaatctgcgcgaaatcggtgcggcgattcagaaatttgtcgaagcagaa ggcttctccgtcgttcgtgaatattgcggacacggtattggtcgcggcttccatgaagaaccgcaggtgctgcactatga ctcccgtgaaaccaacgtcgtactgaaacctgggatgacgttcaccatcgagccaatggtcaacgcgggtaaaaaagaga tccgcaccatgaaagatggctggacggtaaaaaccaaagatcgcagcttgtctgcacaatatgagcatactattgtggtg actgataacggctgcgaaattctgacgctacgcaaggatgacaccatcccggcgataatctcgcacgacgaataa SEQ ID NO. 2 : Amino acid sequence of MAP protein

Sequence ID no. 2: MAP aminoacid sequence (275 aminoacid seq)

MAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAVSACLGY HGYPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKDGFHGDTSKMFIVGKPTIMGER LCRITQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREYCGHGIGRGFHEEPQVLH YDSRETNVVLKPGMTFTIEPMVNAGKKEIRTMKDGWTVKTKDRSLSAQYEHTIVVTDNGC EILTLRKDDTIPAIISHDE HHHHHHDDDDK

DETAILED DESCRIPTION OF THE I NVENTION

As used herein, "heterologous protein" or "protein of interest" refers generally to peptides and proteins exogenous i.e. foreign to the E. coli cells. Examples of the protein includes molecules such as, colony stimulating factors (CSFs), for example M-CSF, GM-CSF, and G-CSF; growth hormone, including human growth hormone; interferon such as interferon- alpha, -beta, and -gamma; interleukins (ILs), such as IL-2, IL-1 1 , IL-1 RA; reteplase, staphylokinase, Steptokinase DPP-4, DPP-8, PTH, PDGFAA, PDGFAB, PDGFBB and fragments of any of the above-listed polypeptides.

In an embodiment of the invention there is provided a fusion DNA comprising MAP protein and an enzymatic cleavage site. In another embodiment there is provided a fusion DNA comprising MAP protein and an enzymatic cleavage site wherein the fusion tag increases the solubility of the protein of interest.

In another embodiment there is provided a vector comprising the fusion DNA comprising MAP protein an enzymatic cleavage site, protein of interest.

In an embodiment there is provided a process for producing heterologous peptides or proteins in a soluble and stable form in E. coli cells. Specifically, the invention provides a method for producing a protein of interest in the soluble form comprising:

a) obtaining a fusion DNA comprising a first DNA encoding MAP protein, Enterokinase site 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.

In another embodiment any commercial vector and promoter may be used such as pET21 a, pBAD24, pQE, etc. The preferred vector is pET21 a.

In an embodiment of the invention any enzymatic cleavage site may be used such as enterokinase, TEV protease, Factor Xa and thrombin etc. The preferred enzyme cleavage site is of enterokinase .

The process of the invention provides to express protein of interest as soluble entities in E coli cells which are reported to be usually expressed in insoluble form (inclusion bodies). To obtain the protein of interest from the inclusion bodies, processes like harsh denaturation conditions, refolding etc are required which are tedious, time-consuming, not universal and expensive and not applicable for large scale manufacturing scale operations. In another embodiment the protein of interest which may be expressed by the present invention are cytokines, hormones, thrombolytic agents, cleavage enzymes, enzymes related to glycosylation or deglycosylation like PNGaseF, endo HF, , restriction enzymes and the like. Cytokines includes Interleukins, Interferons, Growth factors, Colony Stimulating factors and the like. Hormones includes PTH, FSH, GH, LH and the like.

In an embodiment of the invention the present invention may be used to express the protein of interest which require external supply of rare codons for example interferon or proteins which have strong secondary mRNA structure for example IL-2. In an embodiment of the invention MAP protein is used as a fusion tag to express proteins of interest in E. coli cells is less in size(MAP is 39 kDa) as compared with other tags like NusA tag, the molar ration of the fusion tag versus protein of interest will be low which in turn results in higher yield of the proteins of interest after separation from the fusion partner.

In an embodiment of the invention the gene of interest was amplified from a template and digested with BamHI/Hindlll and cloned into pETMAPF vector. The resultant clone was screened with colony PCR and confirmed by restriction digestion. The clones were then introduced into DE3 cells and were used for expression analysis using 1 mM IPTG. The induced cultures were pelleted, lysed by homogenizer and soluble and insoluble fraction was separated by centrifugation. The samples were then analysed on SDS-PAGE. The fusion expressed as a soluble protein was treated with enterokinase to get protein of interest.

In a further embodiment of the invention provided for a fusion protein DNA and the process to expresses the proteins in soluble fraction when expressed as N terminal fusion protein.

The fusion DNA comprises of MAP protein with an enterokinase site (EK) at C terminus and a 6X His tag at its N terminus. EK site ensures the generation of authentic N terminus of protein of interest after cleavage of the fusion protein with enterokinase, while the N terminus 6X His tag aids in purification of the fusion protein through a single step of metal affinity chromatography.

In an embodiment where several molecules ranging from small molecules like human IL-2 (14 kDa) to large molecules like PNGaseF (36 kDa) to a molecule having several disulphide linkages like reteplase (9 disulphides) have been demonstrated to be successfully expressed as MAP fusion proteins using the process of the invention. The proteins of the invention fractionated into the soluble fraction of the E. coli cells. Thus providing for soluble fractions of the protein of interest expressed by the process of the invention.

In another embodiment of the invention the protein of interest is obtained in correctly folded form like PNGaseF protein was found to be active after enterokinase digestion. The Enterokinase site may be optionally replaced by other cleavage enzymes like TEV protease, Factor Xa and thrombin etc. Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description of preferred embodiments thereof.

Example 1 : Cloning, expression and purification of MAP molecule MAP molecule was amplified using following gene specific primers using E. coli cells genomic DNA. The primers used were 5' CCG CCG GAA TTC CAT ATG GCT ATC TCA ATC AAG ACC CCA GAA 3' and reverse primer which contains histidine 6x tag, 5' CCG CCG GAA TTC AAG CTT TTA ATG ATG ATG ATG ATG ATG TTC GTC GTG CGA GAT TAT CGC 3' and annealing temperature used was 50 °C for first five cycles and 60 °C for remaining 25 cycles.. The amplified MAP gene was digested with Ndel and Hindi 11 and cloned in pET21 a vector.

Plasmid DNA was isolated from the cultures and restriction analysis was done to confirm the release of insert by Nde1/Hindlll digestion. The resultant clones were designated as pETMAP.

The clones were then introduced into DE3 cell line and were used for expression analysis. The BL21 (DE3) pETMAP clones were inoculated into 50 ml LB amp and kept in orbital shaker at 37 °C. The cultures were induced with 1 mM IPTG and after 4 h were pelleted at 8000 rpm for 10 min. The pellets obtained were then lysed with homogenizer and soluble and insoluble fractions were separated by centrifuging at 13000 rpm for 20 min. The protein expression was analyzed on SDS- PAGE gel.

The MAP protein is expressed completely in soluble fraction. The MAP protein was further purified using Nickel NTA agarose and the bound protein eluted from column was pure shows the purified MAP protein. example 2: cloning and construction of map fusion vector

MAP gene was amplified using E. coli cells gene as template with following primers as forward primer 5' CCG CCG GAA TTC CAT ATG GCT ATC TCA ATC AAG ACC CCA GAA 3' and reverse primer 5' CCG CCG GAA TTC AAG CTT TTA ATG ATG ATG ATG ATG ATG TTC GTC GTG CGA GAT TAT CGC 3'. The amplified PCR product was cloned into pET21 a vector at Ndel BamHI sites. The clones were confirmed by restriction digestion. The resultant vector was designated as pETMAPF and vector map is given in Figure 1 .

The clones were introduced into BL21 A1 cell line and were inoculated in 50 ml LB amp and induced after 1 h with 13 mM arabinose and 1 % lactose for 4h. The cell were pelleted and disrupted in a cell disruptor. The lysed cell suspension was centrifuged at 13000 rpm for 20 min and the soluble and insoluble fractions were separated. The protein expression was analyzed on a 13 % SDS- PAGE.

It was found that MAP protein was completely restricted to the soluble fraction of the E. coli cytoplasm.

This vector was designated as pMAPF and contains MCS site with BamHI, Hindi 11, EcoRI, Sacl, Sail, Xhol etc sites. This vector was used for cloning of different genes to get soluble expressions for the respective proteins. The vector was designed in such a way that after enterokinase digestion, the protein of interest will have authentic amino acid.

Example 3: Cloning and expression of reteplase molecule as MAP fusion

Reteplase (RTP) is a protein having high molecular weight 39 kDa containing 9 disulphide bonds. RTP molecule was amplified from a synthetic template and digested with BamHI and Hindlll and cloned into pETMAPF vector (MAP without stop codon). The primers used for amplification were as follows forward primer 5' CCG CCG GGA TCC GAT GAT GAT GAT AAA TCT TAC CAA GGC AAC AGC GAT TGC 3'and reverse primer 5' CCG GAA TTC AAG CTT TTA CGG TCG CAT GTT GTC ACG AAT CCA 3' and annealing temperatures were 50 °C for first five cycles and 60 °C for remaining 25 cycles. So the resultant clone contains MAPRTP fusion with enterokinase site before N terminus of RTP. The clone was confirmed with restriction digestion.

The clones were then introduced into a BL21 (DE3) cell line and were used for expression analysis. The BL21 (DE3) MAPRTP clone was inoculated into 250 ml LB medium with ampicillin (amp) and kept in orbital shaker at 37 °C. The cultures were induced with 1 mM IPTG and after 4 h were pelleted at 8000 rpm for 10 min. The pellets were suspended in 10 mM Tris CI obtained was then lysed with homogenizer and soluble and insoluble fractions were separated by centrifuging at 13000 rpm for 20 min. The protein expression was analyzed on SDS- PAGE. The MAP RTP fusion protein was expressed as a soluble protein. The RTP molecule was expressed as a fusion of MAP protein was tested for activity using calorimetric assay and blood clot lysis assay and was found to be active.

When MAPRTP fusion lysate was incubated with enterokinase, the fusion protein was digested into two fragments MAP and RTP proteins.

Example 4: Cloning and expression of PNGase F molecule as MAP fusion

PNgase F gene (1065 bp) is 39 kDa protein is used in removal of carbohydrate moeitis from glycoproteins. The PNGase F gene was subcloned into pETMAPF vector at EcoRI site from a synthetic template. The resultant clone contains MAPPNGaseF fusion with enterokinase site before N terminus. The clones were confirmed with restriction digestion. The clones were introduced into DE3 cell line and were used form expression analysis. The DE3 MAP PNGaseF clones was inoculated into 250 ml LB amp and after 2 h was induced with 1 mM IPTG and incubated in orbital shaker for 16h at 18 °C. The cells were pelleted and lysed with homogenizer and soluble and insoluble fractions were separated by centrifuging at 13000 rpm for 20 min. The MAP-PNGaseF fusion protein seen as 69 kDa band was expressed in the soluble fraction as seen on SDS-PAGE gel.

The BL21 (DE3) MAP-PNGaseF lysate was incubated with enterokinase and both MAP and PNGaseF proteins were observed in SDS-PAGE gel.

Example 5: Activity analysis of PNGaseF derived from MAP-PNGaseF clone:

The PNGaseF protein was digested and purified from E coli MAP-PNGaseF soluble fraction. The purified protein was further analyzed for activity with help of two glycosylated proteins eg. Staphylokinase and enterokinase derived from Pichia pastoris.

These two glycosylated proteins were incubated with purified PNFaseF at 37^<Ό for 2 hours and samples were run on the 13.5% SDS -Page gel. The glycosylated Staphylokinase protein runs at 18 kDa, after deglycosylation it was running at 14 kDa in the both inhouse PNGaseF and Commercial PNGaseF treated samples. Similarly in the case of glycosylated Enterokinase, after deglycosylation the size of enterokinase was reduced from 43 kDA to 33 kDa. Example 6: Cloning and expression of IL-2 molecule as a MAP fusion

Interleukin 2 (IL-2) is a cytokine protein of 14 kDa size and used in therapeutic applications. IL-2 gene was amplified from a synthetic template using forward primer 5' CCG CCG GGA TCC GAT GAT GAT GAT AAA CCT ACT TCA AGT TCT ACA AAG 3' and 5' CCG GAA TCC AAG CTT TCA AGT CAG TGT TGA GAT GAT GCT 3' digested with BamHI Hindlll and cloned into pETMAPF vector. The resultant clone contains MAPIL- 2 fusion with enterokinase site before N terminus. The clones were confirmed with restriction digestion.

The clones were introduced into DE3 cell line and were used form expression analysis. The BL21 (DE3) MAP IL-2 clones was inoculated into 250 ml LB amp and after 2 h was induced with 1 mM IPTG and incubated in orbital shaker for 16h at 18 °C. The cells were pelleted and lysed with homogenizer and soluble and insoluble fractions were separated by centrifuging at 13000 rpm for 20 min. The MAPIL-2 fusion protein was expressed in soluble fraction as seen on SDS-PAGE gel.

Example 7: Cloning and expression of IFN molecule as MAP fusion

IFN gene was amplified from a synthetic template using forward primer 5' CCG CCG GGA TCC GAT GAT GAT GAT AAA TGT GAC CTA CCA CAA ACC CAC 3' and reverse primer 5' CCG CCG GAA TTC AAG CTT TTA TCA TTC CTT ACT TCT TAA ACT TTC 3'digested with BamHI Hindlll and cloned into pETMAPF vector. The resultant clone contains MAPIFN fusion with enterokinase site before N terminus. The clones were confirmed with restriction digestion.

The clones were introduced into BL21 (DE3) cell line and were used form expression analysis. The BL21 (DE3) MAP IFN clones was inoculated into 250 ml LB amp and after 2 h was induced with 1 mM IPTG and incubated in orbital shaker for 16h at 18 °C. The cells were pelleted and lysed with homogenizer and soluble and insoluble fractions were separated by centrifuging at 13000 rpm for 20 min. The MAPIFN fusion protein was expressed in soluble fraction as seen on SDS-PAGE gel. The IFN protein was released after enterokinase digestion of DE3 MAPIFN lysate. The most important advantage with this vector is tRNA's for rare codons are not required since the expression of MAPIFN fusion protein was achieved without supplementation of the specific rare codon tRNA's. Example 8: Cloning and expression of EGF as MAP fusion

Epidermal Growth factor (EGF) gene was amplified from a synthetic template using forward primer 5' CCG CCG GGA TCC GAT GAT GAT GAT AAA AAT AGT GAC TCT GAA TGT CCC CTG 3' and reverse primer 5' CCG CCG AAG CTT TAC GTA TTA GTG CAG TTC CCA CCA CTT CAG 3' and digested with BamHI and Hindi 11 and cloned into pETMAPF vector. The resultant clone contains MAPEGF fusion with enterokinase site before N terminus. The clones were confirmed with restriction digestion.

The clones were introduced into BL21 (DE3) cell line and were used form expression analysis. The DE3 MAPEGF clones was inoculated into 250 ml LB amp and after 2 h was induced with 1 mM IPTG and incubated in orbital shaker for 16h at 18 °C. The cells were pelleted and lysed with homogenizer and soluble and insoluble fractions were separated by centrifuging at 13000 rpm for 20 min. The MAP-EGF fusion protein was expressed in soluble fraction.

Example 9: Cloning and expression of human enterokinase as MAP fusion

Human enterokinase gene was amplified from a synthetic template and digested with EcoRI Hindi 11 and cloned into pETMAPF vector. The resultant clone contains MAPEK fusion with enterokinase site before N terminus. The clones were confirmed with restriction digestion.

The clones were introduced into BL21 (DE3) cell line and were used form expression analysis. The BL21 (DE3) MAP EK clones was inoculated into 250 ml LB amp and after 2 h was induced with 1 mM IPTG and incubated in orbital shaker for 16h at 18 °C. The cells were pelleted and lysed with homogenizer and soluble and insoluble fractions were separated by centrifuging at 13000 rpm for 20 min. The MAPEK fusion protein was expressed in soluble fraction.

When the soluble MAP-EK fusion protein was purified with nickel column, the MAPEK fusion protein was self cleaved into MAP protein and EK protein.

Example 10: Cloning and expression of IFN, IL-2, RTP and EK gene in pET21a vector without fusion tags:

IFN, IL-2, RTP and EK gene were amplified and cloned at pET21 a at Ndel Hindi II site to test the expression of the proteins without any tags. RTP and EK proteins were expressed in BL 21 DE3 cell line were fractionated in insoluble fraction. IFN required rare codons to express and were expressed only in insoluble fraction of BL21 DE3 codon plus cell line. Also, IL-2 was expressed at very low levels in insoluble fraction BL21 DE3 cell line.

Example 11 : Cloning and expression of Ranibizumab heavy and light chain as MAP fusion:

Ranibizumab heavy and light gene was amplified from a synthetic template and digested with BamHI and Hindlll and cloned into pETMAPF vector. The resultant clones contain MAPHC and MAPLC fusion with enterokinase site before N terminus. The clones were confirmed with restriction digestion.

The clones were introduced into BL (DE3) cell line and were used form expression analysis. The BL21 (DE3) MAPHC and MAPLC clones were inoculated into 30 ml LB amp and after 1 h were induced with 0.5 mM IPTG and incubated in orbital shaker for 4 h at 18 °C. The cells were pelleted and lysed with homogenizer and soluble and insoluble fractions were separated by centrifuging at 13000 rpm for 20 min. The MAPHC and MAPLC fusion protein was expressed in soluble fraction.

Hence, MAP protein was successfully used as a fusion tag to fractionate heavy and light chains of Ranibizumab into soluble fraction in E coli.

Claims

CLAIMS:

1 . A process for the preparation of heterologous protein in bacterial cells comprising the steps of:

a) Preparing a fusion DNA comprising a DNA fragment encoding MAP protein and a DNA fragment fused in frame encoding the heterologous protein of interest, b) Insertion of the fusion DNA of step a in a vector,

c) Cloning of the vector comprising the fusion DNA of step a,

d) Expressing the fusion protein in bacterial cells and extraction thereof from bacterial cells,

e) Obtaining protein of interest from fusion protein,

f) Optionally purifying the protein of interest.

2. The process of claim 1 , wherein the heterologous protein of interest is selected from the group comprising of monoclonal antibodies, cytokines, growth stimulating factors, hormones, interferons, interleukins and enzymes.

3. The process of claim 1 , wherein the heterologous protein of interest is selected from the group comprising of ranibizumab, parathyroid hormone (1 -34), parathyroid hormone (1 - 84), reteplase, interferon, IL-2, IL-3, IL-4, IL-5, IL-6, IL-1 1 and GCSF.

4. The process of claim 1 , wherein the bacterial cells are E. coli cells.

5. The process of claim 1 , wherein the fusion 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, size exclusion chromatography.

6. A fusion DNA comprising a DNA fragment encoding MAP protein and a DNA fragment fused in frame encoding the heterologous protein of interest.

7. The fusion DNA of claim 6, further comprises an enterokinase cleavage site.

8. The fusion protein of claim 7, is cleaved at enterokinase site of the fusion protein.

9. The MAP protein of claim 7, having nucleotide sequence of SEQ ID 1.

10. The MAP protein of claim 7, having amino acid sequence of SEQ ID 2.