WO2013076657A1 - Map fusion protein - Google Patents

Map fusion protein Download PDF

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WO2013076657A1
WO2013076657A1 PCT/IB2012/056583 IB2012056583W WO2013076657A1 WO 2013076657 A1 WO2013076657 A1 WO 2013076657A1 IB 2012056583 W IB2012056583 W IB 2012056583W WO 2013076657 A1 WO2013076657 A1 WO 2013076657A1
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protein
fusion
map
interest
dna
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PCT/IB2012/056583
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English (en)
French (fr)
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Shardul Salunkhe
Sriram Padmanabhan
Jitendra Kumar
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Lupin Limited
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Priority to IN935MUN2014 priority Critical patent/IN2014MN00935A/en
Publication of WO2013076657A1 publication Critical patent/WO2013076657A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand

Definitions

  • 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.
  • MAP methionine aminopeptidase
  • E. coli 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.
  • 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.
  • heterologous proteins are expressed in E coli in high levels, recombinant proteins are frequently expressed in E.
  • inclusion bodies insoluble protein aggregates termed as "inclusion bodies".
  • small proteins >30 kDa
  • proteins ⁇ 30 kDa or proteins that have complex secondary or tertiary structures are typically insoluble and are predominantly found in inclusion bodies.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the invention relates to a process for the production of the heterologous protein in E. coli cells, the process comprises of:
  • the invention is related to a fusion protein DNA sequence comprising MAP protein sequence fused to a DNA of the heterologous protein of interest.
  • the invention provides a process for the production of heterologous protein as a soluble fusion protein using a MAP protein as a fusion tag.
  • 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
  • Sequence ID no. 2 MAP aminoacid sequence (275 aminoacid seq)
  • heterologous protein or “protein of interest” refers generally to peptides and proteins exogenous i.e. foreign to the E. coli cells.
  • 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.
  • CSFs colony stimulating factors
  • M-CSF M-CSF
  • GM-CSF GM-CSF
  • G-CSF growth hormone
  • growth hormone including human growth hormone
  • interferon such as interferon- alpha
  • a fusion DNA comprising MAP protein and an enzymatic cleavage site.
  • a fusion DNA comprising MAP protein and an enzymatic cleavage site wherein the fusion tag increases the solubility of the protein of interest.
  • a vector comprising the fusion DNA comprising MAP protein an enzymatic cleavage site, protein of interest.
  • a process for producing heterologous peptides or proteins in a soluble and stable form in E. coli cells provides a method for producing a protein of interest in the soluble form comprising:
  • any commercial vector and promoter may be used such as pET21 a, pBAD24, pQE, etc.
  • the preferred vector is pET21 a.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 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.
  • 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.
  • Reteplase 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.
  • 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.
  • BL21 (DE3) MAP-PNGaseF lysate was incubated with enterokinase and both MAP and PNGaseF proteins were observed in SDS-PAGE gel.
  • 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.
  • glycosylated proteins were incubated with purified PNFaseF at 37 ⁇ ⁇ 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.
  • 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 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 GAT AAA CCT ACT TCA AGT TCT ACA AAG 3' and 5' CCG GAA TCC AAG CTT TCA AGT CAG TGT TGA 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • MAP protein was successfully used as a fusion tag to fractionate heavy and light chains of Ranibizumab into soluble fraction in E coli.

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Cited By (1)

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WO2018211529A1 (en) 2017-05-19 2018-11-22 Council Of Scientific & Industrial Research A method for producing refolded recombinant humanized ranibizumab

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WO2018211529A1 (en) 2017-05-19 2018-11-22 Council Of Scientific & Industrial Research A method for producing refolded recombinant humanized ranibizumab
US11524996B2 (en) 2017-05-19 2022-12-13 Council Of Scientific & Industrial Research Method for producing refolded recombinant humanized ranibizumab
US11578122B2 (en) 2017-05-19 2023-02-14 Council Of Scientific & Industrial Research IPTG-free induction process for expression of biosimilar rHu Ranibizumab antibody fragment using E. coli

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