Field of the Invention
The present invention provides a method of obtaining a N-terminal methionine free polypeptides in E.coli by constructing a plasmid to express a Ubiquitin like protein SMT3-polypeptide fusion protein in the E.coli cytoplasm, which is cleaved by the co- expressed ULP1 protease. The SMT3-polypeptide fusion protein of the instant invention is specifically a SMT3-Streptokinase, a SMT3-Streptokinase fusion protein.
Background and prior art of the Invention
Streptokinase is a thrombolytic drug approved for use in acute myocardial infarction and thromboembolism. Streptokinase acts on plasminogen to convert it into plasmin which in turn degrades fibrin into soluble products. Streptokinase C (SKC) is synthesized as a precursor protein of 440 amino acids from which a 26 amino acid signal sequence is cleaved off before secretion from hemolytic Streptococci. Both native (non-recombinant) and recombinant SKC (rSKC) are commercially available.
In two recent studies it was reported that, of the sixteen SKC preparations available in the market, only three showed potency fulfilling the criteria established by European Pharmacopoeia (Longstaff C, Thelwell C, and Whitton C., J. Thromb. Haemost3: 1092-1093, 2005 and Hermentin P, Cuesta-Linker T, Weisse, Schmidt K-H, Knorst M, Scheid M and Thimme M, European Heart Journal, 26:933-940, 2005). Discrepancies were observed in potency of many marketed rSKC as measured by in vitro assays as compared to a reference standard. Among the three rSKC samples included in the study, two showed potency ranging from 20.8% to 37.2% of the label claim, while a third rSKC preparation from China showed abnormal reaction kinetics and hence potency could not be determined at all. The N-termini of rSKC was found to be variable. While one rSKC preparation from Cuba showed approximately 50% of molecules with correct N-termini beginning with isoleucine, while the other 50% still had intact methionine at N-termini. An rSKC preparation from China had 6 extra amino acids at the N-terminus, presumably from the signal sequence. The third rSKC from India gave two sequences, both of which had no relationship to SKC N-terminus. Native SKC has the following amino acid sequence at N-terminus: IAGPEWLLDRPSVNH.
Currently SKC potency evaluation is done by two in-vitro assays - one in the absence of fibrin using a chromogenic substrate and another using fibrin as a substrate for clot- lysis where the activity is declared in international reference units. The value obtained by these two assays is identical when native streptokinase is tested. If the manufacturer of the recombinant SKC choose only one method (either chromogenic or clot-lysis assay) to determine potency, it is possible that the potency values obtained could be either be overestimated or underestimated. This under or over estimation has dramatic consequences during treatment. If the dose administered is lower than recommended, then reperfusion rate could be lower. Where as a high dose of streptokinase administered could cause increase intracranial hemorrhage.
The N-terminus of Streptokinase plays a key role in Plasminogen activation during fibrin clot lysis Mundada LV, Prorok M, DeFord ME, Figuera M, Castellino FJ, Fay WPJ Biol Chem. 2003 Jul 4;278(27):24421-7. We have observed variation in the activity is up to two fold between the two assays for recombinant strepokinases with or without the N-terminal methionine. Thus there is a need to make a good quality recombinant SKC which is methionine free at its N-terminus, herein after called Met0 SKC, whose potency as measured by both chromogenic and clot-lysis assay are identical and comparable to properties of native SKC. The method used to manufacture Met0 SKC should be also simple, efficient and commercially viable.
When recombinant proteins are expressed in E.coli, it is necessary to have ATG codon, encoding Methionine for initiation of protein synthesis. When heterologus proteins are expressed in E.coli, the N-terminal methionine may be removed in vivo, by methionine amino peptidase encoded by the genome of E.coli. In some cases, either due to primary or secondary structure, or inaccessibility of the N-terminus, or simply due to over expression, the N-terminal methionine fails to be removed. One method to solve this problem is to employ a tag at the N-terminus of the recombinant protein with suitable cleavage site to construct a fusion protein. Methods for producing a recombinant protein with an N- or C-terminal tag are well known in the art. For therapeutic applications, the tag is removed from the purified recombinant protein by enzymatic cleavage in vitro, to generate the authentic N-terminus bearing protein. Many tags are available to the practitioners of art. An example of such a strategy is adding
Glutathione S-transferase (GST) tag ending in C-terminus with amino acids Ile- Glu/Asp-Gly-Arg, which is cleaved specifically by Factor Xa protease, resulting in a recombinant protein with authentic N-terminus. This method is cumbersome as Factor Xa protease has to be produced separately and purified before its use in vitro for fusion protein cleavage. Also, many of the proteases also cleave the fusion protein at various non-canonical sites reducing overall yield of recombinant protein.
The table below summarizes the current state of art, wherein only enterokinase, Factor Xa protease and intein tags allow cleavage in a specific manner, without leaving behind extra amino acids at the N-terminus.
Excision site I Cleavage Enzyme/ Self- Cleavage Comments
Asp-Asp-Asp-Asp-Lysj Enterokinase *The site will not cleave if followed by a proline residue. Secondary cleavage sites at other basic residues, depending on conformation of protein substrate. Active from pH 4.5 to 9.5 and between 4°C and 45°C.
Ile-Glu/Asp-Gly-Argj Factor Xa protease Will not cleave if followed by proline and arginine. Secondary cleavage sites following Gly-Arg sequences.
Leu-Val-Pro-Arg|Gly-Ser Thrombin Secondary cleavage sites. Biotinylated form available for removal with immobilized streptavidin.
Glu-Asn-Leu-Tyr-Phe- GlnlGly TEV protease Seven-residue recognition site, making it a highly site-specific protease. Active over a wide range of temperatures. Protease available as a His-tag fusion, allowing for protease removal after recombinant protein cleavage.
Leu-Glu-Val-Leu-Phe- Gln|Gly-Pro PreScission™ protease Genetically engineered form of human rhinovirus 3C protease with a GST fusion, allowing for facile cleavage and purification of GST-tagged proteins along with protease removal after recombinant protein cleavage. Enables low-temperature cleavage of fusion proteins containing the eight-residue recognition sequence.
Specific intein-encoded sequences Intein 1 & intein 2 Uses self-cleavable affinity tags. Even after cleavage unnatural termini are present on the protein of interest.
Signal sequences Signal peptidases Cleavage of leader sequence concomitant with protein export from the cytoplasm.
♦LaVallie, E.R., McCoy, J.M., Smith, D.B. & Riggs, P. (1994). Enzymatic and chemical cleavage of fusion proteins. In Current Protocols in Molecular Biology, pp. 16.4.5-16.4.17, John Wiley and Sons, Inc, New York, NY.

The reason for the ever increasing number of tags made available for various N- terminus cleavages is due to the fact that it is not possible to predict whether a particular tag will provide the desired result. Two common reasons for using a tag are to improve the expression of a recombinant protein or make the expressed recombinant protein soluble. Often, recombinant heterologus proteins expressed in E.coli cytoplasm become insoluble forming the so called "inclusion bodies" and the use of tag may make the protein soluble at least partially. In other cases the tag may make the purification process simpler. For example adding a His tag to the expressed recombinant protein, allows simple and rapid purification using metal affinity chromatography step. One could also combine two tags, for example, His tag (comprising of six or ten Histidine residues) could be fused to another tag (for example, SMT3) which could make the recombinant protein more soluble. The advantage of His tag is easy purification from cytoplasm, using metal affinity chromatography.
A second reason for adding tags to N-terminus is to make the proteins stable and prevent its degradation by proteases in E.coli cytoplasm. Small peptides/proteins with low molecular weight (< 10 kD) are often degraded rapidly when expressed by themselves in E.coli. For example, small peptides like GLP-1, insulin, etc have been shown to be stable only when they are expressed along with a tag at N-terminus.
Small Ubiquitin related Modifier protein (SUMO), is a protein which is covalently attached post translationally to N-terminus of various proteins. SMT3 from Saccharomyces cerevisiae is an example of Small Ubiquitin like protein. Ubiquitin and Ubiquitin like proteins play an important role in various cell functions (nuclear metabolism, cell proliferation, autophagy, etc.) by modulating protein structure and function. ULP1 protease releases SMT3 tag from proteins to which it is attached. Interestingly prokaryote like E.coli has neither SMT3 nor ULP1 protease gene in its genome. Hence use of SMT3 tag in prokaryotes is much more convenient. There are also no proteases described in E.coli which could cleave SMT3. Also, ULP1 protease, due to its high specificity can not cleave any E.coli protein and is thus not toxic to E.coli. Hence use of SMT3 tag in E.coli is much easier as compared to eukaryote like yeasts, in which there is a protease which cleaves SMT3 tag in the middle, after which due to alteration in three dimensional structure of the SMT3 protein; the C-terminal half SMT3 attached to a protein of interest fails to be cleaved by ULP1 protease, unless one adds back N-terminal half SUMO to the cleavage reaction.
Ubiquitin-like-speciflc protease 1 (ULP1) is deubiquitinating enzyme which cleaves the C-termini of SMT3 and deconjugate SMT3 from the side chains of lysines (Li, S.J. and Hochstrasser, M. (1999) Nature 398, 246-251). Three-dimensional structure of a complex between SMT3 and ULP1 show that certain surface features of SMT3 are recognized by ULP1 that make the interaction specific (Mossessova, E. and Lima, C.D. (2000) Mol. Cell. 5, 865-876). The catalytic domain (amino acids 403-621) of ULP1 is sufficient for deconjugation of SMT3 from fusion proteins and has been show to be functional when expressed in E.coli (Mossessova, E. and Lima, C.D. (2000) Mol. Cell. 5, 865-876).
Objects of the present invention:
The principal object of the present invention is to produce N-terminal methionine free polypeptides in microbial host cells.
Another object of the present invention is to produce N-terminal methionine free polypeptides in bacterial and fungal host cells.
Yet another object of the present invention is to produce N-terminal methionine free streptokinase in E.coli host cells.
Still another object of the present invention is to develop a method for the making of N-terminal methionine free polypeptides in microbial host cells. Still another object of the present invention is to develop a method for the making of N-terminal methionine free streptokinase in E.coli host cells.
Still another object of the present invention is to arrive at pure form of recombinant N- terminal methionine free streptokinase (Met0 SKC).
Still another object of the present invention is to prepare a pharmaceutical composition comprising streptokinase (Met0 SKC) along with one or more pharmaceutical^ acceptable excipients.
Still another object of the present invention is to prepare a pharmaceutical composition comprising Met0 hGH along with one or more pharmaceutically acceptable excipients.
Statement of Invention:
A method of making N-terminal methionine free polypeptide in a recombinant microbial host cell comprising: (i) preparing an expression construct containing a DNA encoding a polypeptide of interest; (ii) preparing an expression construct containing a DNA encoding a protease enzyme (iii) cloning both the expression constructs of (i) and (ii) into the host cell to obtain transformant; (iv) fermentatng the transformant and inducing co-expression; and (v) obtaining the N-terminal methionine free polypeptide; a method of making N-terminal methionine free streptokinase in E.coli host comprising: (i) preparing an expression construct containing a DNA encoding a SUMO- streptokinase fusion protein; (ii) preparing an expression construct containing a DNA encoding a Ulpl protease; (iii) cloning both the expression construct of (i) and (ii) into an E.coli host; (iv) fermentation of the transformants and inducing co- expression; and (v) obtaining the N-terminal methionine free streptokinase by lysing the cells and purifying the protein; a substantially pure recombinant N-terminal methionine free streptokinase (Met0 SKC); a pharmaceutical composition comprising streptokinase (Met0 SKC) which is substantially free of Met-SKC along with one or more pharmaceutically acceptable excipients; and a pharmaceutical composition comprising Met0 hGH which is substantially free of N-terminal methionine-hGH along with one or more pharmaceutically acceptable excipients.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig.l. Map of the plasmid with SKC coding sequence driven by a T7 promoter sequence
Fig.2. SDS-PAGE analysis of total cell lysate from induced and uninduced cultures of E.coli BL21 (DE3) transformed with pET SKC plasmid.
Fig.3. Map of the plasmid with mutated version of E.coli MAP coding sequence driven by native promoter.
Fig.4. SDS-PAGE analysis of total cell lysate from induced and uninduced cultures of E.coli BL21 (DE3) transformed with pET ULP1 plasmid.
Fig.5. Schematic diagram showing method of construction of plasmid for co-expression
of Saccharomyces cerevisiae ULP1 in E.coli
Fig.6. Schematic representation of SMT3 and SKC gene fusion
Fig.7 is a map of the plasmid with SMT3-SKC fusion coding sequence driven by a T7 promoter sequence
Fig.8. SDS-PAGE analysis of total cell lysates from induced and uninduced cultures of E.coli BL21 (DE3) co-transformed with pET SKC and pTrcULPl/pACYC plasmids. Fig:9 Illustration explaining the soluble chromogen assay and clot lysis assay. Description of Certain Preferred Embodiments of the Invention The present invention is in relation to a method of making N-terminal methionine free polypeptide in a recombinant microbial host cell comprising:
(i) preparing an expression construct containing a DNA encoding a polypeptide of interest;
(ii) preparing an expression construct containing a DNA encoding a protease enzyme
(iii) cloning both the expression constructs of (i) and (ii) into the host cell to obtain transformant;
(iv) fermentatng the transformant and inducing co-expression; and
(v) obtaining the N-terminal methionine free polypeptide.
Another embodiment of the present invention wherein the microbial host cell is selected from a group comprising bacteria and fungi.
Yet another embodiment of the present invention wherein N-terminal methionine free polypeptide is btained either by secrection or by lysing the cells and purifying the protein.
Still another embodiment of the present invention wherein the polypeptide of interest is a hormone, an enzyme or a therapeutic protein.
Still another embodiment of the present invention wherein the hormone is a growth hormone, luteinising hormone, or a natriuretic peptide
Still another embodiment of the present invention wherein the therapeutic enzyme is streptokinase
Still another embodiment of the present invention wherein the growth hormone is hGH The present invention is in relation to a method of making N-terminal methionine free streptokinase in E.coli host comprising:
(i) preparing an expression construct containing a DNA encoding a SUMO- streptokinase fusion protein;
(ii) preparing an expression construct containing a DNA encoding a Ulpl protease;
(iii) cloning both the expression construct of (i) and (ii) into an E.coli host;
(iv) fermentation of the transformants and inducing co-expression; and
(v) obtaining the N-terminal methionine free streptokinase by lysing the cells and purifying the protein.
Another embodiment of the present invention wherein the E.coli host is a BL21 or BL26 strain
Yet another embodiment of the present invention wherein the expression construct is a pET vector containing a DNA encoding SMT3-Streptokinase fusion protein. Still yet another embodiment of the present information, wherein the expression construct is a pACYC184 vector containing a DNA encoding Ulpl protease operationally linked to a promoter.
Still yet another embodiment of the present information wherein the DNA encoding Ulpl protease operationally linked to a promoter is integrated into E.coli genome. Still yet another embodiment of the present information wherein N-terminal methionine free streptokinase is Met0 SKC of Seq. ID no. 17, with potency identical to native SKC. The present invention is in relation to a substantially pure recombinant N-terminal methionine free streptokinase (Met0 SKC)
The present invention is in relation to a pharmaceutical composition comprising streptokinase (Met0 SKC) which is substantially free of Met-SKC along with one or more pharmaceutically acceptable excipient
The present invention is in relation to a pharmaceutical composition comprising Met0 hGH which is substantially free of N-terminal methionine-hGH along with one or more pharmaceutically acceptable excipients.
Another embodiment of the present invention, wherein the pharmaceutical excipients are selected from a group comprising granulating agents, binding agents, lubricating agents, disintegrating agents, sweetening agents, coloring agents, flavoring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents and spheronization agents.
Recombinant SKC when expressed in E.coli without a tag at the N-terminus results in a mixture of molecules with and without Methionine. The physicochemical properties of the rSKC molecules with and without Methionine are very similar and are hence difficult to separate by simple techniques. In the current invention, we have genetically engineered a SMT3 tag at the N-terminus of SKC to generate fusion protein, which is cleaved by the co-expressed ULP1 protease resulting in Met0 SKC with correct N- terminus, matching that of native streptokinase. A vector has been developed in which PCR amplified SMT3 coding sequence has been fused in frame with PCR amplified coding sequence of mature SKC (Met0 SKC) and cloned into a vector in which the fusion protein sequence is operationally linked to phage T7 promoter. This recombinant vector is cloned to express the recombinant SMT3-Met° SKC fusion protein in a genetically engineered E.coli host like BL21 (DE3) or BL26 (DE3) strains, upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). The recombinant SMT3 -SKC fusion protein is expressed into the E.coli cytoplasm and is cleaved by the by co-expressed ULP1 in vivo. The ULP1 protease very specifically recognizes the three dimensional features of SMT3 protein, in addition to Gly-Gly sequence, in the C- terminus and cleaves the SMT3 to release Met0 SKC. This simplifies the method of cleavage as opposed to in vitro cleavage which requires a second fermentation to produce ULP1 protease. The ULP1 protease is expressed in the same cell by a compatible, low copy number recombinant plasmid vector into which PCR amplified coding sequence of ULP1 protease is cloned operationally linked to tac promoter which is also inducible by IPTG.
The instant invention allows a simple method of producing Met0 SKC in large quantities using recombinant techniques for use in pharmaceutical compositions. The alternate methods described in literature involve over expression of E.coli gene coding for methionine amino peptidase (MAP). MAP over expression does not always help in removal of methionine from the N-terminus for various reasons like the nature of penultimate amino acid, secondary structure of the protein near the N-terminus, etc. It has been previously reported that if the amino acid next to initiator methionine is Ala, Gly, Pro and Ser, it is easily cleaved by MAP. If the penultimate amino acid is lie, Val, Cys or Thr, then there is variably in cleavage and removal of methionine. Finally, if the penultimate amino acid is Lys, Arg, Leu, Asp, Asn, Phe, Met, Trp, Tyr, Glu, Gin or His, the initiator methionine is not removed. (Ben-Bassat A, Bioprocess Technology 12:147-159, 1991). In the case of SKC, penultimate amino acid is Isoleucine and hence we also found variability in removal of methionine by MAP
A recent report (Liao YD, Jeng JC, Wang CF, Wang SC, Chang ST., Protein Science. 13:1802-1810, 2004, US Patent 7109015) suggested that site-directed mutagenesis of three residues in E.coli methionine amino peptidase substrate binding pocket (Y168G, M206T, Q233G) allowed the removal methionine from proteins even if the penultimate residues were Met, His, Asp, Asn, Glu, Gin, Leu, lie, Tyr, and Trp. However, in the case of SKC, neither over expression of the native MAP nor over expression of the mutated E.coli MAP (with Y168G, M206T & Q233G amino acid substitutions) failed to remove methionine from N-terminus of Met SKC completely, even though penultimate residue is lie. SKC there fore appears to be unique protein, removal of methionine from which appears to be a challenge. Because of these constraints, we sought an alternate method in which close to 100% of the recombinant SKC molecules produced would be methionine free.
SMT3 coding sequence was PCR amplified from genomic DNA of Saccharomyces cerevisiae. SKC mature form coding nucleic acid fragment was amplified from the genomic DNA of Streptococcus equisimilis. By employing overlapping primers, the C- terminal end of SMT3 (ending in amino acids Gly-Gly) was fused in frame to N- terminal of Met0 SKC (beginning with amino acids Ile-Ala). This fused PCR product was cloned into pET vector using which recombinant proteins could be made by induction with IPTG after transformation into appropriate commercially available genetically engineered E.coli host like BL21 (DE3), into whose genome lacUV5 promoter driven T7 phage polymerase coding sequence has been integrated (Novagen, Madison, USA). PCR technique was used to amplify ULP1 protease gene from Saccharomyces cerevisiae genome and cloned into a low copy number vector (pACYC) vector. The expression of ULP1 was driven by tac promoter, which is also induced by IPTG. An alternative method of co-expression of ULP1 protease by integration of the ULP1 gene into E.coli genome is also possible. Preferably the ULP1 gene could be targeted to msbB gene coding sequence, the disruption of which could cause a reduction in the levels of endotoxin from the E.coli host, helping in improving the quality of therapeutic protein by reduction of endotoxin impurity.
The technology of the instant Application is further elaborated with the help of following examples. However, the examples should not be construed to limit the scope of the invention. Example 1
Cloning and Expression of rSKC in E.coli.
Preparation of replicative plasmid for intracellular production of Streptokinase in E.coli
Amplification of SKC gene:
The source of DNA for mature Streptokinase encoding gene was the genomic DNA isolated from Streptococcus sp. strain BICC #7869 (MTCC 389). Genomic DNA was isolated using a commercially available (Qiagen) kit following manufacturer's instructions. The following primers were used for the amplification of the SKC gene.
SKC FP1: 5' CAT ATG ATT GCT GGA CCT GAG TGG CTG CTA 3' (Seq. ID#1) SKC RP1: 5' CGG GAT CCT TAT TTG TCG TTA GGG TTT ATC AGG 3' (Seq. ID#2).
Proof reading Pwo polymerase (Roche) was used for PCR and the SKC gene was amplified. The amplified product was electrophoresed on 1% agarose gel and was purified using the gel extraction kit (Qiagen). The gel purified product was modified to have dATP overhangs and this product was ligated to pTZ57R/T vector (MBI Fermentas).The ligation reaction was carried out at 16°C overnight. The ligation mix was transformed to chemically competent E.coli DH5a cells and plated on LB Ampicillin 100 jig/ml plates. The transformants were screened by isolating plasmid from 1 ml of culture by mini lysis method and was digested with restriction enzymes BamHI and Xbal (NEB) to release the insert. The digestion was carried out at 37°C for 2 hours. One positive clone was selected and inoculated to LB medium containing Ampicillin 100 |xg/ml and plasmid was isolated by Qiagen mini prep kit and the plasmid was sequenced to confirm the presence of the gene and any possible mutations. The sequence confirmed plasmid was then used for further sub-cloning.
Sub cloning SKC coding DNA fragment to pET vector: The plasmid which has the SKC gene in pTZ57R/T vector was digested with the restriction enzymes Ndel and BamHI and electrophoresed on 1% agarose gel. The insert released was excised and gel eluted and ligated to pET vector which was also digested with the same enzymes. The ligation was done using the enzyme T4 DNA ligase (NEB) and ligation was carried out at 16 °C overnight. The ligation mix is used to transform competent E.coli DH5 alpha cells and the transformants were selected on LB plates with 100 ng/ml Ampicillin. The transformants are screened for the presence of the SKC gene. The correct clone (Fig. 1) was mapped and confirmed and this plasmid is used for transforming competent E.coli BL26 (DE3) host.
Expression in E.coli BL26 (DE3) host: The verified plasmid clone was transformed to E.coli BL26 (DE3) host and the transformants were selected on Ampicillin (100 fig/ml) plates. A few transformants were grown in LB Ampicillin (100|ig/ml) liquid media and when the OD at 600nm is -0.6 to 0.8 the cells are induced with IPTG (1 mM). The induction is carried out overnight at 30 °C and the cells were pelleted and resuspended in buffer and sonicated to lyse the cells. The samples were loaded on SDS-PAGE. The results are shown in Fig. 2. A good level of induction was observed. Recombinant SKC was purified and sequencing of the product showed that only 40% of the rSKC molecules were Met0 SKC.
Example 2.
Over expression of E.coli MAP in E.coli expressing SKC
The main goal of this experiment was to determine if over expression of MAP in the same cell as that expressing rSKC would help in efficient removal of N-terminal methionine.
E.coli MAP gene was amplified from BL21 (DE3) host genomic DNA using the following primers (Seq. ID# 3 to Seq. ID# 10) to generate a mutated version which has been reported to remove N-terminal methionine more efficiently from proteins with lie as the penultimate amino acid (Liao YD, Jeng JC, Wang CF, Wang SC, Chang ST., Protein Science. 13:1802-1810, 2004, US Patent 7109015). The mutations introduced were 168Y, 206M, 233Q.
Y168GFP 5TTCGTGAAGGCTGCGGACACGGTATTGGTCGCGG3' Seq. ID# 3
Y168GRP 5'GTCCGCAGCCTTCACGAACGACGGAGAAGCCTTC3' Seq. ID# 4
M206TFP 5'CGAGCCAACCGTCAACGCGGGTAAAAAAGAGATC3' Seq. ID# 5
M206TRP 5'CGTTGACGGTTGGCTCGATGGTGAACGTCATCCC3' Seq. ID# 6
Q233GFP 5'GTCTGCAGGCTATGAGCATACTATTGTGGTGACTG3' Seq. ID# 7
Q233GRP 5'GCTCATAGCCTGCAGACAAGCTGCGATCTTTGG3' Seq. ID# 8
MetAPFP 5'GGATCCGACGTCGAATTTTCTATTA3' Seq. ID# 9
MetAPRP 5' TCGGGTAT AGCT ATG A A AGC AGCTG3' Seq. ID# 10

The primer positions have been represented in the following figure.
MetAPFP Y168GPP |M206TFP Q233GFP
► ► *
M + < 4
|Y168GKP |M20CTRP |Q233GRP | MetAPRP

The source of DNA for Methionine amino peptidase (MAP) was E.coli genomic DNA from BL21 (DE3) strain which was isolated by routine protocols (Molecular Cloning: A Laboratory Manual By Joseph Sambrook, David W Russell, 2001, CSHL Press). Four separate PCR reactions were performed using E.coli genomic DNA as template and the proof-reading polymerase, Pwo polymerase with various primer pairs as outlined in the following table:
Primer pairs Size of amplified product
MetAPFP/ Y168GRP 510bp
Y168GFP/M206TRP 132bp
M206TFP/Q233GRP 96bp
Q233GFP/MetAPRP 310bp

The four PCR reactions were run on 1.2% agarose gel for 1 hr at 100 mV. Individually the amplified fragments were excised from the gel and purified using Qiagen gel extraction kit. To get full length MAP gene (1048kb), all the four fragments were used as template using MetAPFP & MetAPRP primers. Proof reading Pwo polymerase (Roche) was used for PCR and the full length MAP gene was amplified. The amplified product was electrophoresed on 1% agarose gel and was purified using the gel extraction kit (Qiagen). The gel purified product was modified to have dATP overhangs and this product was ligated to pTZ57R/T vector (MBI Fermentas).The ligation reaction was carried out at 16°C overnight. The ligation mix was transformed to chemically competent E.coli DH5a cells, plated on LB Ampicillin (100|ig/ml) plates incubated at 37 °C, overnight. The transformants were screened by isolating plasmid from 1 ml of culture by mini lysis method and was digested with restriction enzymes BamHI and Xbal (NEB) to release the insert. The digestion was carried out at 37°C for 2 hours. One positive clone was selected and inoculated to LB broth containing Ampicillin (100 (ig/ml), incubated at 37 °C shaker incubator, overnight. Plasmid was isolated by Qiagen mini prep kit and the plasmid was sequenced to confirm the presence of the gene and mutations made at three positions.. The sequence confirmed plasmid was then used for further sub-cloning.
Sub-cloning MAP (Sea. ID #18) gene to pACYC vector: The plasmid with the MAP gene in pTZ57R/T vector was digested with the restriction enzymes Sail followed by BamHI and was electrophoresed on 1% agarose gel. The insert released was excised from the gel, eluted and ligated to pACYC vector which was also digested with the same enzymes sequentially. The ligation was done using the enzyme T4 DNA ligase (NEB) and ligation wais carried out at 16 °C overnight. The ligation mix was used to transform competent E.coli DH5 cells and the transformants were selected on LB plates with Chloramphenicol (15|ig/ml). The transformants were screened for the presence of the MAP gene. The correct clone was mapped and confirmed (Fig. 3).
Expression in E.coli BL26 (DE3) host: The verified plasmid clone was co- transformed along with pET SKC vector to E.coli BL26 (DE3) host and the transformants were selected on Ampicillin (100 ng/ml) and Chloramphenicol (15 |ig/m)l plates. A few transformants were grown in LB broth with Ampicillin (100ng/ml) and Chloramphenicol (15|ig/ml). When the OD at 600 nm was -0.6 to 0.8 the cells were induced with IPTG (1 mM). The induction was carried out overnight at 30 °C and the cells were pelleted and resuspended in buffer and sonicated to lyse the cells. The samples were loaded on SDS-PAGE.
After studying the expression of rSKC (Seq. ID # 17) and purification, the results indicated that there was no improvement in efficiency of removal of methionine from N-terminus (data not shown). Approximately 40%-60% of the rSKC molecules still had methionine at their N-terminus.
Example 3:
Cloning and Expression of SMT3-SKC fusion in E.coli
The genomic DNA of Saccharomyces cerevisiae strain (BICC# 7806) was isolated using standard techniques. This was used as the template and a PCR reaction was carried out using the primers SMT FP1 (5' CATATGGGTCATCACCATCATCATCACGGGTCGG ACTCAGAAGTC 3', Seq. ID# 11) and SMT RP1 (5 'CTCAGGTCCAGCAATACCTC CAATCTGTTCGCGGTG 3', Seq. ID# 12), the SMT-3 gene was amplified. The reaction mix was electrophoresed on 1.2% agarose gel. The amplified product was checked for the size and was gel eluted and was used as the template for overlapping PCR. In a similar way using the genomic DNA of the strain BICC 7869 and the primers SMT-SKC FPI (5' GAACAGATTGAAGGTATTGCTGGACCTGAGTGGCTG 3', Seq. ID# 13) and SMT-SKC RP1 (5'CGG GAT CCT TAT TTG TCG TTA GGG TTT ATC AGG 3', Seq. ID# 14) the SKC gene was amplified by PCR using proof reading polymerase. This fragment was also gel eluted and used as template for overlapping PCR. By mixing both the purified PCR products and using them as the template, PCR was performed to get the full length overlapped product. This is electrophoresed on 1% agarose gel and the product is gel eluted. This is then ligated to pTZ57R/T vector by creating dATP overhangs and the ligation was carried out at 16°C overnight. The ligation mix is then transformed to competent E.coli DH5a cells and the mix was plated on LB containing Ampicillin (lOOjig/ml) plates and the plates were incubated at 37°C incubator overnight. The transformants which appear were screened for the presence of the gene and this plasmid was purified by using Qiagen mini prep kit. This plasmid was sequenced to confirm the overlap and for mutations. The confirmed plasmid was used for further experiments.
This plasmid was digested with the restriction enzymes Ndel and BamHI (NEB) and the reaction mix was incubated at 37°C for 2 hours. The reaction was terminated by heat inactivation of the enzymes and it was electrophoresed on 1% agarose gel. The insert released was gel eluted and was used for ligation with the vector. The vector is prepared by digesting pET vector with the enzymes Ndel and BamHI and processed in a similar way as done to the insert. Then a ligation reaction was set using T4 DNA ligase (NEB) and the reaction was carried out at 16°C overnight. The ligation mix was then transformed to competent E.coli DH5a cells and then selected on LB Kanamycin (25|ig/ml) plates. The transformants were screened for the presence of the gene and the plasmid was isolated from the correct clone using Qiagen Mini prep kit. The plasmid was confirmed by restriction analysis and was then used for expression studies.
Schematic representation of SMT3 AND SKC genefusion is illustrated in Fig: 6. The plasmid containing SMT3-SKC fused gene (Fig: 7) product in pET vector was used to transform competent E.coli BL21 (DE3) cells. The transformants were selected on LB containing Kanamycin (25|xg/ml) plates. A few colonies were inoculated to 100 ml LB containing Kanamycin (25|ag/ml) and were grown to OD 600 of -0.7-0.8. A small sample was removed as control and the rest of the culture was induced with IPTG to a final concentration of 1 mM. The induction was performed overnight at 30°C with shaking at 120 rpm. The cells were then pelleted and resuspended in buffer and sonicated to lyse the cells and the samples were analyzed on 10% SDS-PAGE.
Example 4:
Cloning and expression of Ulpl protease in E.coli
Using the primers Ulpl FP (Ndel) 5' CAT ATG CTT GTT CCT GAA TTA AAT GAA AAA GAC G 3' (Seq. ID# 15) and Ulpl RP (Xhol) 5' CTC GAG TTT TAA AGC GTC GGT TAA AAT C 3' (Seq. ID# 16), the ULP1 (Seq. Id # 19) gene was amplified by PCR using Saccharomyces cerevisiae genomic DNA as the template (BICC# 7806) and proof reading Pwo polymerase (Roche) enzyme. The PCR product was gel purified and cloned to pTZ57R/T vector (MBI Fermentas) after creating dATP overhangs. The ligation was carried over night at 16 °C. The ligation mix was transformed to competent E.coli DH5a cells and selected on LB Ampicillin (100|ig/ml) plates. The colonies were screened by PCR using Ml3 forward and Ml3 reverse primers and gene specific forward and reverse primers. One correct clone was selected and plasmid from this clone was isolated by using Qiagen mini prep method. This clone was sequenced to confirm the presence of the gene and any possible mutations
Results of sequencing of the PCR product showed three mutations. All three mutations were silent mutations (Asp413 GAC to GAT, Ser473 TCA to TCG, Glu554 GAG to GAA). The correct confirmed clone was digested with restriction enzymes Ndel and Xhol (NEB) and the insert released was gel purified and ligated to the pET vector which was also digested with Ndel and Xhol restriction enzymes. The ligation was done using the enzyme T4 DNA ligase (NEB) and the reaction was carried out at 16°C overnight. The ligation mix was then transformed to competent E.coli DH5a cells by heat shock method. The transformation mix was then plated on LB plates carrying Kanamycin (25ng/ml) and the plates were incubated at 37°C overnight. The Kanamycin resistant transformants were screened for the presence of the gene and the correct clone was inoculated to LB medium with Kanamycin (25|ig/ml) and plasmid was isolated by using Qiagen mini prep kit.
After confirming the authenticity of the plasmid by restriction digestions, it was transformed to chemically competent E.coli BL21 (DE3) competent cells and was plated on LB Kanamycin (25 |ig/ml) plates.The plates were incubated at 37 °C overnight. The Kanamycin resistant transformants were checked for protein production. A few Kanamycin resistant transformants were inoculated to 100 ml of liquid LB broth containing Kanamycin (25 ^g/ml) and this was grown at 37°C till the OD at 600 nm reached 0.8. At this stage the culture was induced and a small amount of sample was removed as contol. Induction was done with IPTG (1 mM) and the culture was further grown at 30 °C overnight. After overnight IPTG induction, the OD at 600 nm was checked and the cells were pelleted by centrifugation at 6000 rpm for 6 minutes. The cells were then resuspended in a buffer (100 mM Tris buffer, pH 8.0 with 0.1M NaCl) and sonicated using Branson sonifier till the OD dropped to 1/5 -1/8 of the initial OD. The soluble fraction was loaded on to Ni-NTA agarose matrix.(Qiagen) which was equilibrated with the same buffer. The matrix was washed with the same buffer and the protein was eluted from the matrix using the same buffer supplemented with 250 mM
Imidazole. The results are shown in Fig. 5. A good level of induction (and Ulpl production) was observed and the His-tagged Ulpl was purified on Ni-NTA column chromatography.
Example 5:
Co-expression of SMT3-SKC fusion protein and ULP1 protease in E.coli (Sea Id # 201
Sub cloning of Ulpl to pTre99A
The Ulpl protease encoding DNA fragment which was cloned to pET vector was digested with the restriction enzymes Ndel and BsrBI (NEB) and the digestion is carried out by incubating the reaction mix at 37 °C. The reaction mix was then heated to high temperature to inactivate the enzymes and electrophoresed in l%agarose gel. The fragment released is then gel eluted and is ligated to pTrc99A vector which was also digested with the enzymes Ndel and Smal and was treated in similar manner. The ligation reaction was carried out using T4 DNA ligase (NEB) and the reaction was carried out at 16 °C overnight. The ligated sample was then used to transform chemically competent E.coli DH5a cells and plated on LB Ampicillin (lOOg/ml) plates. The transformants which were resistant to Ampicillin (lOOg/ml) were screened for the presence of the insert by PCR using the gene specific primers. One correct clone was identified and used for further experiments.
Sub cloning Ulpl coding sequence to pACYC184 vector:
The Ulpl gene which was cloned to pTrc99A vector was digested with enzymes PvuII and BamHI (NEB) and the digestion was carried out at 37 °C for 2 hours. The reaction was stopped by heat inactivating the enzymes and the mix was electrophoresed on 1% agarose gel. The insert released was gel purified and used for ligation with the vector. The pACYC 184 vector was prepared by digesting with enzymes EcoRV and BamHI.The restriction enzyme digested pACYC 184 vector was purified in a similar manner (Fig: 8). The insert and the vector were ligated using T4 DNA ligase. The ligated sample was transformed to competent DH5a cells and plated on LB agar plates containing Chloramphenicol (lO^g/ml). The plates were incubated at 37 °C overnight.

The Chloramphenicol resistant transformants were screened and the correct clone was confirmed by restriction analysis. This plasmid was used for further experiments. This pACYC184 vector with insert of Ulpl gene in was co-transformed along with the plasmid harboring SMT3-SKC gene to chemically competent E.coli BL21 (DE3) host and plated on LB plates with Chloramphenicol (10 g/ml) & Kanamycin (25 g/ml).The plates were incubated at 37°C overnight. The transformants which appeared were resistant to both the antibiotics indicating the presence of both the plasmids. A few transformants were inoculated to 100 ml of LB medium containing both the antibiotics and the flask was incubated on a shaker at 37 °C. When the OD at 600 nm reached -0.7-0.8, a small sample was removed as control and the rest of the culture was induced with IPTG to a final concentration of 1 mM. The culture was next incubated at 30 °C with shaking at 120 rpm overnight. The OD at 600 nm of the culture at the end of induction was checked and the cells were pelleted by centrifugation. The cell pellet was resuspended in a buffer containing 20 mM Tris, pH 8.0 and 100 mM NaCl. After resuspension the OD at 600 nm was checked and the volume was adjusted to -20-25 OD and the cells were sonicated using a cell disruptor, keeping on ice so that the temperature does not increase. The samples were loaded on 10% SDS-PAGE gel (Fig. 4). The cleavage was very efficient when both the protease and SMT3-SKC (Seq Id #21) were co expressed in the same host. The SKC was purified from lysed E.coli cells by standard purification methods. N-terminus of all the purified rSKC molecules was confirmed to be free of methionine.
Met SKC and Met free SKC were purified and analyzed for biological activity in solution chromogenic assay and Fibrin clear clot assay (Illustrated in Fig:9). The third international reference standard for Streptokinase (NIBSC Batch # 00/464 derived from Native Streptokinase) was used to calculate the relative potency and activity. The ratio of the activity for the two streptokinase preparations by these two methods was calculated and is presented in the table below
Batch number Met SKC Met free SKC

1.06
2.0
1.01
2.5
0.91
1.7
Batch #1 Batch #2 Batch #3
Seq. ID#1
CAT ATG ATT GCT GGA CCT GAG TGG CTG CTA Seq. ID#2
CGG GAT CCT TAT TTG TCG TTA GGG TTT ATC AGG Seq. ID#3
TTC GTG AAG GCT GCG GAC ACG GTA TTG GTC GCG G Seq. ID#4
GTCCGCAGCCTTCACGAACGACGGAGAAGCCTTC Seq. ID#5
CGAGCCAACCGTCAACGCGGGTAAAAAAGAGATC Seq. ID#6
CGTTGACGGTTGGCTCGATGGTGAACGTCATCCC Seq. ID#7
GTCTGCAGGCTATGAGCATACTATTGTGGTGACTG Seq. ID#8
GCTCATAGCCTGCAGACAAGCTGCGATCTTTGG Seq. ID#9
GGATCCGACGTCGAATTTTCTATTA Seq. ID#10
TCGGGTATAGCTATGAAAGCAGCTG Seq. ID#11
CATATGGGTCATCACCATCATCATCACGGGTCGGACTCAGAAGTC Seq. ID#12
CTCAGGTCCAGCAATACCTCCAATCTGTTCGCGGTG Seq. ID#13
GAACAGATTGAAGGTATTGCTGGACCTGAGTGGCTG Seq. ID#14
CGG GAT CCT TAT TTG TCG TTA GGG TTT ATC AGG Seq. ID#15
CAT ATG CTT GTT CCT GAA TTA AAT GAA AAA GAC G Seq. ID# 16
CTC GAG TTT TAA AGC GTC GGT TAA AAT C Seq. ID# 17
Amino acid sequence of mature rSKC
1 IAGPEWLLDR PSVNNSLVVS VAGTVEGTNQ DISLKFFEID LTSRPAHGGK 51 TEQGLSPKSK PFATDSGAMS HKLEKADLLK AIQEQLIANV HSNDDYFEVI
101 DFASDATITD RNGKVYFADK DGSVTLPTQP VQEFLLSGHV RVRPYKEKPI 151 QNQAKSVDVE YTVQFTPLNP DDDFRPGLKD TKLLKTLAIG DTITSQELLA 201 QAQSILNKNH PGYTIYERDS SIVTHDNDIF RTILPMDQEF TYRVKNREQA 251 YRINKKSGLN EEINNTDLIS EKYYVLKKGE KPYDPFDRSH LKLFTIKYVD 301 VDTNELLKSE QLLTASERNL DFRDLYDPRD KAKLLYNNLD AFGIMDYTLT 351 GKVEDNHDDT NRIITVYMGK RPEGENASYH LAYDKDRYTE EEREVYSYLR 401 YTGTPIPDNP NDK
Seq. ID# 18
AMINO ACID SEQUENCE OF E.coli Methionine aminopeptidase (MAP) WITH THREE MUTATIONS (Bold and underlined).
1 MAISIKTPEDIEKMRVAGRL AAEVLEMIEP YVKPGVSTGE LDRICNDYIV 51 NEQHAVSACL GYHGYPKSVC ISINEVVCHG IPDDAKLLKD GDIVNIDVTV 101 IKDGFHGDTS KMFIVGKPTI MGERLCRITQ ESLYLALRMV KPGINLREIG 151 AAIQKFVEAE GFSVVREGCG HGIGRGFHEE PQVLHYDSRE TNVVLKPGMT 201 FTIEPTVNAG KKEIRTMKDG WTVKTKDRSL SAGYEHTIVV TDNGCEILTL 251 RKDDTIPAII SHDE
Seq. ID# 19
Nucleotide sequence of the ULP1 PCR product amplified using Saccharomyces cerevisiae genome as template. The PCR product amplified nucleotides encoding amino acids 403-621 of ULP1 protein. The silent nucleotide substitutions are shown in bold and underlined.
1 CATATGCTTG TTCCTGAATT AAATGAAAAA GACGATGATC AAGTACAAAA 51 AGCTTTGGCA TCTAGAGAAA ATACTCAGTT AATGAATAGA GATAATATAG 101 AGATAACAGT ACGTGATTTT AAGACCTTGG CACCACGAAG ATGGCTAAAT 151 GACACTATCA TTGAGTTTTT TATGAAATAC ATTGAAAAAT CTACCCCTAA 201 TACAGTGGCG TTTAATTCGT TTTTCTATAC CAATTTATCA GAAAGGGGTT 251 ATCAAGGCGT CCGGAGGTGG ATGAAGAGAA AGAAGACACA AATTGATAAA 301 CTTGATAAAA TCTTTACACC AATAAATTTG AACCAATCCC ACTGGGCGTT 351 GGGCATAATT GATTTAAAAA AGAAAACTAT AGGTTACGTA GATTCATTAT
401 CGAATGGTCC AAATGCTATG AGTTTCGCTA TACTGACTGA CTTGCAAAAA 451 TATGTTATGG AAGAAAGTAA GCATACAATA GGAGAAGACT TTGATTTGAT 501 TCATTTAGAT TGTCCGCAGC AACCAAATGG CTACGACTGT GGAATATATG 551 TTTGTATGAA TACTCTCTAT GGAAGTGCAG ATGCGCCATT GGATTTTGAT 601 TATAAAGATG CGATTAGGAT GAGAAGATTT ATTGCCCATT TGATTTTAAC 651 CGACGCTTTA AAACTCGAG
Seq. ID# 20
Amino acid sequence of the ULP1 gene fragment expressed in E.coli:
1 MLVPELNEKD DDQVQKALAS RENTQLMNRD NIEITVRDFK TLAPRRWLND 51 TIIEFFMKYI EKSTPNTVAF NSFFYTNLSE RGYQGVRRWM KRKKTQIDKL 101 DKIFTPINLN QSHWALGIID LKKKTIGYVD SLSNGPNAMS FAILTDLQKY 151 VMEESKHTIG EDFDLIHLDC PQQPNGYDCGIYVCMNTLYG SADAPLDFDY 201 KDAIRMRRFI AHLILTDALK LEHHHHHHHH
Seq. ID# 21
Amino acid sequence of SMT3-SKC fusion protein. The sequence of SMT3 is shown in bold letters.
1 MGHHHHHHGS DSEVNQEAKP EVKPEVKPET HINLKVSDGS SEIFFKIKKT 51 TPLRRLMEAF AKRQGKEMDS LRFLYDGIRI QADQAPEDLD MEDNDIIEAH 101 REQIGGIAGP EWLLDRPSVN NSQLVVSVAG TVEGTNQDIS LKFFEIDLTS 151 RPAHGGKTEQ GLSPKSKPFA TDSGAMSHKL EKADLLKAIQ EQLIANVHSN 201 DDYFEVIDFA SDATITDRNG KVYFADKDGS VTLPTQPVQE FLLSGHVRVR 251 PYKEKPIQNQ AKSVDVEYTV QFTPLNPDDD FRPGLKDTKL LKTLAIGDTI 301 TSQELLAQAQ SILNKNHPGY TIYERDSSIV THDNDIFRTI LPMDQEFTYR 351 VKNREQAYRINKKSGLNEEINNTDLISEKY YVLKKGEKPY DPFDRSHLKL 401 FTIKYVDVDT NELLKSEQLL TASERNLDFR DLYDPRDKAK LLYNNLDAFG 451 IMDYTLTGKV EDNHDDTNRIITVYMGKRPE GENASYHLAY DKDRYTEEER 501 EVYSYLRYTG TPIPDNPNDK

We Claim:
1) A method of making N-terminal methionine free polypeptide in a recombinant microbial host cell, said method comprising steps of:
a. preparing an expression construct containing a DNA encoding a SUMO- polypeptide of interest fusion protein;
b. preparing an expression construct containing a DNA encoding a Ulpl protease;
c. cloning both the expression constructs of steps (i) and (ii) into the host cell to obtain transformant;
d. fermenting the transformant and inducing co-expression; and
e. obtaining the N-terminal methionine free polypeptide.
2) The method as claimed in claim 1, wherein the microbial host cell is selected from a group comprising bacteria and fungi.
3) The method as claimed in claim 1, wherein N-terminal methionine free polypeptide is obtained either by secretion or by lysing the cells and purifying the protein.
4) The method as claimed in claim 1, wherein the polypeptide is a hormone, enzyme or a therapeutic protein selected from a group comprising growth hormone, luteinising hormone, natriuretic peptide, streptokinase and human growth hormone.
5) A method of making N-terminal methionine free streptokinase in E. coli host cell, said method comprising steps of:
a. preparing an expression construct containing a DNA encoding a SUMO- streptokinase fusion protein;
b. preparing an expression construct containing a DNA encoding a Ulpl protease;
c. cloning both the expression constructs of steps (i) and (ii) into an E.coli host to obtain transformant;
d. fermenting the transformant and inducing co-expression; and
e. obtaining the N-terminal methionine free streptokinase by lysing the cells and purifying the protein.

6) The method as claimed in claim 5, wherein the E.coli host is a BL21 or BL26 strain.
7) The method as claimed in claim 5 (i), wherein the expression construct is a pET vector containing a DNA encoding SMT3-Streptokinase fusion protein.
8) The method as claimed in claim 5 (ii), wherein the expression construct is a pACYC184 vector containing a DNA encoding Ulpl protease operationally linked to a promoter.
9) The method as claimed in claim 5, wherein the DNA encoding Ulpl protease operationally linked to a promoter is integrated into E.coli genome.
Dated this 29th day of November 2006

RAVI BHOLA Of K&S Partners Agent for the Applicant
10) The method as claimed in claim 5, wherein N-terminal methionine free streptokinase is Met0 SKC of Seq. ID no. 17, with potency identical to native
SKC.
11) A method of making N-terminal methionine free polypeptide in a recombinant microbial host cell and a method of making N-terminal methionine free streptokinase in E.coli host cell are substantially as herein described along with accompanied examples and drawings.