COMPLETE SPECIFICATION
(See section 10, rule 13)

“PROCESS FOR PRODUCTION OF RECOMBINANT HUMAN BNP AND RECOVERY OF THE SAME”

VIRCHOW BIOTECH PRIVATE LIMITED of Plot No 4, S.V.Co-op Indl Est. IDA Jeedimetla, Hyderabad -500 055, India.

The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF INVENTION:
The present invention relates to the field of biotechnology more specifically to the protein production.

BACKGROUND AND PRIOR ART:
For the production of peptides and proteins there are several methods and technologies available, such as extraction, production by rDNA technology, production in cell free expression systems, production in transgenic animals, production in plants, production by chemical synthesis and by enzyme technology using proteolytic enzymes under conditions of displacement of the equilibrium of the reaction towards the formation of peptide bond. The size of the molecule determines the technology most suitable for its production.

Recombinant expression is the preferred mode for synthesis of proteins or peptides. The choice of an expression system for the high-level production of recombinant proteins depends on many factors such as cell growth characteristics, expression levels, intracellular and extracellular expression, posttranslational modifications, and biological activity of the protein of interest, as well as regulatory issues in the production of therapeutic proteins. In addition, the selection of a particular expression system requires a cost breakdown in terms of process, design, and other economic considerations. However, bacterial systems remain most attractive due to low cost, high productivity, and rapid use. Particularly, overexpression of target genes as heterologous proteins in Escherichia coli as a host is often the method of choice, because of extensive knowledge of E. coli genetics, availability of versatile vector systems and host strains, ease of use, low costs and high expression levels.

The production of smaller peptides by expression of peptide encoding DNA in a recombinant host cell such as E.coli is commonly encountered by the problem of enzymatic degradation of the expressed peptide with in the host cell, resulting in partial or complete loss of the peptide. The problem is overcome by expressing the peptides as fusion proteins within the host cell. The fusion protein is formed as insoluble inclusion bodies within the cell, within which the peptide is protected from degradation by proteolytic enzymes.
The fusion protein formed as inclusion bodies is normally solubilized by chaotropic agents or detergents for the recovery of the protein of interest. The said recovery process involves multiple steps such as solubilization (which results in denaturation), refolding and cleavage. The denatured fusion protein is refolded before cleavage with enzyme protease. Though the native structure of fusion protein is not required, the fusion protein is refolded in the enzyme activity buffer for cleavage. In the process of refolding and removal of chaotropes the fusion protein may attain different conformation leading to inefficient cleaving of fusion protein leading low yield of final peptide.

Solubilization of inclusion bodies with low concentration of ionic detergents and cleavage of fusion protein in the presence detergents without refolding the fusion protein can be considered as revolutionary approach as the cumbersome processing steps involved in processing the fusion protein is minimized.

Post solubilization the peptide separation from the fusion sequence may be accomplished by placing a sequence of amino acids at the junction of the fusion partner and peptide, which are specifically recognized and cleaved under appropriate conditions such as chemical cleavage or enzymatic cleavage. Cleavage by chemical methods usually requires harsh reaction conditions that may damage or structurally modify the target peptides in addition to low yields and specificity.

Enzymatic cleavage can be accomplished by placing specific amino acids residues in-between the fusion partner sequence and the peptide sequence, wherein the enzyme protease is substrate specifically may recognize and cleaves at the c-terminus of the last amino acid residue of cleavage site, there by producing native peptide.

Brain natriuretic peptide or BNP is the second member of the natriuretic peptide family originally isolated from the porcine brain. Its highest tissue concentrations were found in the heart, where it works as a cardiac hormone. Endogenous hBNP peptide is synthesized in vivo as an 108 amino acid prohormone termed proBNP that is proteolytically cleaved to release the 32-amino acid C-terminal BNP peptide. B-type natriuretic peptide exerts its biological activities via a membrane-bound guanylyl cyclase receptor termed natriuretic peptide receptor A (NPRA).Binding of BNP to this receptor on endothelial cells and vascular smooth muscle cells results in the cellular synthesis of cyclic guanosine monophosphate (cGMP), which mediates the vascular effects of this peptide. Systemic infusion of a recombinant human brain natriuretic peptide in patients with congestive heart failure results in beneficial hemodynamic actions, including arterial and venous dilatation, enhanced sodium excretion, and suppression of the renin–angiotensin– aldosterone and sympathetic nervous systems accordingly, it is useful in the treatment of congestive heart failure.

Thus, the present invention aims at an efficient method for the production of recombinant human BNP (rhBNP) a physiologically active polypeptide in the form of inclusion bodies and recovery of the same in a single step without the involvement of any chaotropic agents.

OBJECT OF THE INVENTION:
The object of the present invention is to develop a process for an efficient production of recombinant human BNP (rhBNP) a physiologically active polypeptide in the form of inclusion bodies and recovery of the same in a single step.

BRIEF DESCRIPTION OF DRAWINGS:
Figure 1 shows the amino acid sequence of a modified N-terminal growth hormone on the lower line and the wild-type N-terminal on the upper line used as a fusion partner to produce b-type natriuretic peptide.
Figure 2 represents the construction scheme of pGH
Figure 3 represents the construction scheme of pGFB-1
Figure 4 depicts the electrophoretic pattern of the fusion protein before and after solubilization. 13.5% SDS-PAGE analysis of the expression, purification of GH-hBNP fusion protein and pure rhBNP. Lane 1, molecular weight marker; lane 2, 15μg of un-induced total cell lysate; lane 3, 15 μg of induced total cell lysate; lane 4, 10 μg 0.3% SDS solubilized GH-hBNP fusion protein from inclusion body; lane 5, 5μg un-cleaved GH-hBNP fusion protein; lane 6, 5μg cleaved fusion protein; lane 7, 3μg RPC purified pure rhBNP fraction.
Figure 5 represents the chromatogram of the purified rhBNP
Figure 6 is a representation of comparison of rhBNP with standard hBNP.
Figure 7 is a graphical representation of the the biological activity of rhBNP and hBNP.

DETAILED DESCRIPTION OF THE INVENTION:
Accordingly, an object of the present invention is to develop a process for an efficient production of recombinant human BNP (rhBNP) a physiologically active polypeptide in the form of inclusion bodies and recovery of the same.
Further, the process of production of the desired gene product comprises by joining the fusion partner (growth hormone) or containing at least an N-terminal portion of a growth hormone to a DNA sequence coding for the desired gene (human BNP) by placing a linker sequence (Ile-Glu-Gly-Arg) at the junction of the fusion partner and peptide, which are specifically recognized and cleaved under appropriate conditions such as enzymatic cleavage.

Further, the fusion partner is also selected with consideration to promote the formation of fusion protein as inclusion bodies. The selection of an appropriate fusion partner for this purpose will depend, in part, upon the nature of the peptide being produced. Preferably, the fusion partner contains at least about 70 amino acid residues. While there is no upper limit on the number of amino acid residues in the fusion partner, it is preferred not to exceed about 90, since a larger the size of fusion partner the ratio of peptide will be less which in turn leads lower yield of desired peptide. It is also preferred to select a fusion partner which is likely to form fusion protein of which 70-80% are inclusion bodies. The fusion partner is such that the fusion protein contains a higher proportion of hydrophobic amino acid residues, which promote the formation of inclusion bodies.

Preferably, the fusion partner is selected with out any cysteine residues or other wise the cysteine residue is modified to amino acid coding for serine ,lysine , proline. A particularly preferred fusion partner of the invention contains at least an N-terminal portion of a growth hormone. The amino acid cysteine at position 54 was replaced with serine in growth hormone.
The process wherein the desired peptide is linked at its N-terminal to a linker peptide, where it joins the C-terminal end of the fusion partner.

The fusion protein is expressed in a host cell using known techniques available to a person skilled in the art of recombinant DNA production. Any suitable host cell known to be useful for the expression of proteins by recombinant DNA methods may be employed, including prokaryotic and eukaryotic host cells and cell lines. E. coli is a preferred host cell. The host cell contains an expression vector which encodes the fusion protein under the control of a regulatory sequence which is capable of directing its expression in the host, as well as an origin of replication that is functional in the host cell. The vector may contain other DNA sequences conventionally employed in recombinant DNA technology such as sequences encoding selectable markers.

The host cell containing the expression vector is grown and the fusion protein expressed under appropriate conditions. The conditions for growth of the host cell and expression of the fusion protein will vary depending on various factors such as the host cell employed, the promoter and the particular fusion protein being expressed. Those skilled in the art are capable of determining the appropriate conditions for the particular host/vector system employed.

For, extraction of inclusion bodies the cell paste is diluted in volumes of lysis buffer and cells were lysed through a dynamill l. Cell lysate is then further diluted in buffer and centrifuged to recover the inclusion bodies.

The inclusion bodies are then subjected to solublization under pH range from 6.8-9.0 but preferably from 7.5 to 8.0 with ionic detergent sodium dodecyl sulphate (SDS) at a concentration 0.1-1.0 %. The preferred concentration range of SDS is 0.3-0.8 % for maximum solublization upto 90%. In this solubilization step the fusion protein not only gets solublized but in the process the fusion protein purity is improved upto 70%. Low concentration of detergent will not interfere with the enzymatic reaction where as the denatured conformation of fusion has more accessibility to cleavage site rather than in refolded fusion protein. The enzyme cleavage is performed in the presence of SDS at a concentration range of 0.3-0.6%. The final preferred concentration of SDS is below 0.3%.

The fusion protein thus obtained is cleaved using a bovine serine protease at 250C. The bovine Factor Xa (serine protease) specifically recognizes and cleaves at the C-terminal of linker site and release the rhBNP without any N-terminal modification. The digestion showed greater than 90% cleavage resulting in 7 kD GH and 3.4 kD rhBNP.

Further purification steps can be employed using techniques known to those skilled in the art. Such steps may include, for example, HPLC, such as RP-HPLC.
Accordingly, steps in processing of inclusion bodies were made simple wherein the inclusion bodies are solubilized using low concentration of ionic detergent; solubilized fusion protein was diluted and cleaved with enzyme protease. Finally the peptide was separated by reverse phase chromatography.

The process of the invention comprises the following steps:
a. Construction of an expression vector to express the peptide as a fusion protein wherein the peptide N-terminus nucleotide sequence is fused to C-terminus of the fusion partner by a linker sequence wherein (i) the Factor Xa linker sequence C-terminus to the fusion partner with amino acid residues Ile Glu Gly Arg forms a bond with N-terminus of peptide and which is specifically cleaved under enzyme Factor Xa (serine protease) under appropriate buffer conditions. (ii) The fusion protein is expressed as insoluble inclusion bodies in the expression host. (iii) The peptide is basic and highly hydrophilic from that of fusion partner and other host proteins hence peptide is separated by reverse phase chromatography.
b. Cell distruption, separation and recovery of inclusion bodies from the host cell.
c. Solublizing inclusion bodies with lower concentration of 0.3-0.8 percentage of sodium
dodecyl sulphate in the absence of strong chaotrope.
d. Direct cleaving of the solublized fusion protein with Factor Xa without any
process of refolding.
e. recovering the peptide by reverse phase chromatography.

The expression vector is constructed with the small fusion partner preferably with a hydrophobic region of the N-terminal region of the growth hormone. The Vector comprises:
a. a regulatory sequence capable of directing expression of a fusion protein in a host
cell.
b. a DNA sequence encoding N-terminal portion of amino acid sequence of growth
hormone in with the cysteine has been replaced with codons encoding serine, lysine
or proline.
c. a linker sequence encoding isoleucine,glutamic acid, glycine and, Arginine
linked to the C-terminal of truncated growth hormone or N-teminal of the peptide
encoding gene.
e. a DNA sequence encoding the desired peptide and having the enzymatic coding
sequence N-terminally is linked to the downstream to the truncated growth
hormone encoding sequence.

EXAMPLES:
The following examples are for the purpose of illustration of the invention and not intended in any way to limit the scope of the invention.

Example 1:
a) Construction of Expression Vector:
The expression vector is constructed by utilizing a DNA sequence encoding the hydrophobic N-terminal 62 amino acids region of growth hormone. The hydrophobic region in the fusion partner promotes the formation inclusion bodies. The free cysteine in the N-terminal region of fusion partner may be altered to avoid disulfide bond formation with the peptide if, there is any disulfide bridge in the peptide.
In the vector of the invention, the N-terminal growth hormone encoding DNA sequence is under the control of a regulatory sequence which is capable of directing expression of the fusion protein in the host cell. The promoter employed is a T7 promotor or lacZ promoter. A preferred promoter is the E. coli T7 promotor in Escherichia coli. This promoter initiates transcription of the fusion protein encoding DNA sequence in the presence of IPTG in the medium.
b) Synthesis and cloning of a gene encoding a fusion protein to B-type natriuretic peptide:.
The hBNP gene assembly of synthetic gene was performed from component oligo nucleotides. The hBNP nucleotide sequence (GeneBank Accession No BC025785) was designed based on the E.coli rare codon preference. Factor Xa site upstream to BNP encoding sequence was generated by use of oligonucleotide primers.The following primers were used in PCR reaction.
P1 ( 5’- CC GGA TCC ATT GAG GGT CGC AGC CCG AAA ATG- 3’), P2 ( 5’-GTT CAG GGC TCT GGC TGC TTC GGC CGT AAA ATG GAC CGT ATC AGC -3’), P3 ( 5’-TCC TCC AGC GGC CTG GGC TGC AAA GTT CTG CGT CGT CAC TAA TAG- 3’), P4(5’- CCT GAA CCA TTT TCG- 3’), P5 ( 5’-TGG AGG AGC TGA TAC-3’), and P6( 5’- CGG AAT TCC TAT TAG TGA CGA CGC AG- 3’). Equal volumes of primers ~1mg/ml were mixed together and diluted with water to a final concentration of ~1ng/µl for each oligonucleotide. The final concentration 0.2ng/ µl for each oligonucleotide was used along with 20mM Tris-HCl (pH8.8),10mM for KCl, 10mM(NH4)2 SO4 , 6mM MgSO4, 0.1% (V/V) triton –X100, 0.1mg/ml bovine serum albumin,0.2mM each dNTP and 2.5 U Pfu polymerase. The PCR protocol for gene assembly began with an initial denaturation step for 5 min at 94oC, which is followed by 25 cycles of denaturation for 30 s at 94oC, annealing for 60s at 56oC, and extension for 60s at 72oC. It is again followed by an extension step for 10 min at 72oC. hBNP gene was amplified by using 1µl of the mixture resulting from the gene assembly as the template and the outer most oligonucleotides P1 and P6 as primers. The hBNP gene with Factor Xa cleavage sequence was PCR amplified using gene specific primers. The forward primer 5’- CGGATCCATTGAGGGTCGCAGCCCGAAAATG-3’ contains BamH1 and the reverse primer 5’- CGGAATTCCTATTAGTGACGACGCAG- 3’ contains EcoR1 site with Stop codon. The PCR amplification cycles are as follows: Initial denaturation at 94o C 5 min; followed by 25 cycles of denaturation at 94 o C for 30sec, annealing at 56 oC for 1 min, extension at 72 o C for 1 min, and final extention at 72 o C for 10min with a final hold at 15 o C.

Construction of pGFB-1 and expression of hBNP (1-32) fusion protein:
The full length growth hormone gene was used as a template to amplify 186 bps growth hormone using gene specific primers. The primers used for amplification contains Nde1 site in forward direction and BamH1 in reverse primer.
Forward primer F’: 5’- CGCATATGTTCCCAACTATTCCACTGAGT-3’ and Reverse primer R’:5’- CGGGATCCAGGGGTCGGGATACTTTCAGACAAACTCAA-3’. The PCR amplification cycles were as follows: Initial denaturation at 94 oC for 5 min; followed by 30 cycles of denaturation at 94 oC for 30 sec, annealing at 55 oC for 1 min, extension at 72 oC for 1min, and a final extention at 72 oC for 10 min with a final hold at 10oC. The ampicillin resistent plasmid and PCR product was restriction digested with Nde1 and BamH1 endonucleases and ligated by using T4 DNA ligase to yield plasmid pGH. The modified plasmid (pGH) containing 7kDa growth hormone encoding tag under the control of T7 promoter was used to clone hBNP gene. The hBNP PCR product and plasmid pGH were digested individually with restriction endonucleases BamH1 and EcoR1. The digested samples were ran on agarose gel. The agarose gel electrophoresis purified PCR fragment and plasmid pGH were ligated with T4DNA ligase to yield an expression plasmid pGFB-1. The expression construct with hBNP gene was sequenced by Sanger’s Dideoxy method.

Escherichia coli strain was transformed with recombinant plasmid containing the hBNP gene. The expression of the hBNP gene was preferably driven by a T7 promoter, which is regulated by inducing the culture with isopropyl -D- thiogalactopyranoside (IPTG) there by allowing high level of expression of the 7kDa GH-hBNP gene.

Example2:
Production of recombinant human B-type natriuretic peptide:
Fermentation and harvesting:
The seed culture was prepared in LB Media from the Glycerol Stock and incubated for 7-8 hours (O.D of 1.7 to2.3) at 35ºC on rotary shaker. Fermentation was carried out in a Biostat C fermentor (B. BRAUN Germany) in fed-batch mode. Modified LB medium was used for fermentation. The temperature of the fermentation cultures was controlled at 35ºC for a period of 5 hours (O.D of 13 to16) and induced with 0.5 mM IPTG and harvested after 3 hours. The pH was controlled and adjusted to 7.0- 7.2 with 50% Ammonia solution. The rate of agitation was 300 to 700 rpm, and the dissolved oxygen (PO2) was maintained above 40%-50% saturation. Foam formation was suppressed by the addition of sterilized 10% silicon based antifoam. The samples for expression analysis were collected and whole cell lysates were analyzed by SDS PAGE. The culture was centrifuged at 6600g for 10 min, the supernatant was removed and the biomass (525g) was determined by weighing the wet pellet. The cells were frozen immediately at -70ºC until further analysis was performed.

Cell lysis and inclusion bodies (IB) purification:
The cell pellet was thawed on ice, suspended in 787.5ml of Buffer A (50mM Tris and 5mM EDTA pH 8.0) on ice, and homogenized for 2–3 min at maximum speed. Cell disruption was done in dynamill (miltech Switzerland) with 0.5mm of Glass beads up to 80% of the total chamber volume. Cell lysate was passed at a flow rate of 100ml/min which takes about 10 mins for each cycle and the process was optimized to 3 cycles. The lysate at the inlet was maintained at 10-12oC and the sample collected at out let was maintained 15-20oC. The IB fraction was collected by centrifugation at 15,000 g for 20 min at 4oC. The crude IB preparation was initially washed with Buffer A, then with 0.1% deoxycholate (sodium salt) in Buffer A and finally with Buffer A plus 2 M urea. Traces of deoxycholate or urea were removed by a final wash with the Buffer B (20mM Tris pH 7.5). IB pellet was collected by centrifugation at 12,000 g for 20 min at 4 oC and the IB pellet (57g) was stored at -20 oC.

Solubilization of inclusion bodies:
The inclusion body was suspended in 570ml solublization buffer (20mM Tris PH 7.5, 100mM Nacl, 0.3% Sodium dodecyl sulfate (SDS)) and dissolved by stirring overnight at 4°C on a magnetic stirrer. The solution was centrifuged at 12,000g at 4 °C for 10 min. The protein concentration was measured according to the method of Lowry et al. 1975. The supernatant was then diluted with 20mM Tris PH 7.5, to adjust 0.3% SDS to less than 0.1% of final concentration.

Cleavage of GH-hBNP with Factor Xa
The fusion protein (GH-Factor Xa-hBNP) at concentration of 3-5 mg/ml is found to be optimum for cleavage with Factor Xa at 25oC. The bovine Factor Xa (serine protease) specifically recognizes and cleaves at the C-terminal of linker site and release the rhBNP without any N-terminal modification. The digestion showed greater than 90% cleavage results into 7kD GH and 3.4kD hBNP.

Purification of rhBNP by reverse phase chromatography (RPC):
GH-BNP digested mixture was further fractionated on source 30 RPC column to separate pure peptide from other contaminants like residual 11kDa, 7kDa GH and other E.coli host proteins. The peptide was eluted with a linear gradient of acetonitrile. The elution profile showed two peaks. The first (I) peak contained the rhBNP and second peak contained contaminants. rhBNP peak collected was assessed by SDS- PAGE and analytical RP for purity (fig 4). SDS- PAGE analysis showed a single band with both the pools a and b where as the pool a was found to be 60-70 % pure when analyzed by analytical HPLC over C18 column. The 60-70% pure indicating the presence of modified forms like oxidized and deamidated forms. The pool b fractions were highly pure rhBNP which is greater than 99%. These fractions proved to be pure were dialyzed against water injection and next 20mM citric acid buffer pH 4.2. The final yield obtained by this method was approximately 200mg of pure rhBNP /liter cell culture.
Example 3:
Assaying the biological activity of hBNP :
Human aortic endothelial cells at passages 3 through 5 were grown in EGM-2 MV complete medium containing 5% fetal bovine serum (FBS). After confluence, cells were split and cultured in 6-well plates for the experiments. Cells were changed to serum-free medium and pre-incubated with 0.1 mmol/l 3-isobutyl-1-methylxanthine for 1 h, then treated with varying concentrations of human BNP or CHO-derived human proBNP ranging from 0.1 nmol/l to 1,000 nmol/l for 10 min. Cells were lysed with 0.1 mol/l HCl at room temperature for 20 min. The lysates were centrifuged at 6000 g, and the levels of cGMP in the supernatant were measured using a cGMP enzyme immunoassay kit from Assay Designs

Determination of Biological activity of purified recombinant hBNP:
It is suggested that the central ring structure of BNP formed by a disulfide bridge between two cysteine residues is necessary for binding to specific receptors. Hydrolytic disruption of the bridge leads to a loss of biological activity. We have evaluated cGMP production as an index of rhBNP activity in primary human aortic endothelial cells. The graph shows the biological activity at different concentrations of rhBNP in comparison to the standard which was estimated as increased levels of cGMP. As shown in the figure, the rhBNP stimulated intracellular accumulation of cGMP production as the concentration of rhBNP was increased. The calculated EC50 value 35 ± 2.3nmol/l was similar to the standard peptide. Determination of biological activity of purified rhBNP indicated that it has the same potency as the standard.

Example 4:
Construction of pGEX2F-hBNP (GST fusion ) and expression of hBNP(1-32) Fusion protein

The PCR amplified hBNP (1-32) with FXa site encoding gene and the vector pGEX-2F was restriction digested with BamH1 and EcoR1, and the insert was ligated into the vector to generate recombinant plasmid (pGEX-2F/hBNP (1-32)). E. coli strain was transformed with recombinant plasmids containing the hBNP(1-32) gene. The expression of the hBNP (1-32) gene was driven by a Tac promoter, which can be regulated by inducing the culture with isopropyl beta-D-thiogalactopyranoside (IPTG), thereby allowing a high level of expression of the hBNP (1-32) gene as a fusion tag to GST.There by allowing high level of expression of the 30kDa GST-hBNP gene.

Fermentation and harvesting:
The seed culture was prepared in LB Media from the Glycerol Stock and incubated for 7-8 hours (O.D of 1.5 to2.0) at 35ºC on rotary shaker. Fermentation was carried out in a Biostat C fermentor (B. BRAUN Germany) in fed-batch mode. Modified LB medium was used for fermentation. The temperature of the fermentation cultures was controlled at 37ºC for a period of 5 hours (O.D of 14 to16) and induced with 1 mM IPTG and harvested after 3 hours. The pH was controlled and adjusted to 7.0± 0.1 with 50% Ammonia solution. The rate of agitation was 300 to 650rpm, and the dissolved oxygen (PO2) was maintained above 30%-50% saturation. Foam formation was suppressed by the addition of sterilized 10% silicon based antifoam. The culture was centrifuged at 6600g for 10 min, the supernatant was removed and the biomass (240g) 16g/l was determined by weighing the wet pellet. The cells were frozen immediately at -70ºC until further analysis was performed.

Cell lysis and isolation and purification of fusion protein:
The frozen pellet (240g) was thawed on ice and resuspended in 360 ml of phosphate buffered saline (PBS). The pellet was homogenized to get a suspension and homogenate was lysed by using dynamill. The Triton X-100 (1% final) was added to the cell lysate and mixed gently by stirring for 30min at room temperature to aid solubilization of fusion protein. The solution was centrifuged at 18,000 rpm for 30 min at 40C in a Sorval RC-6 with an SV-800 rotor. The clear solution was loaded on to the 500ml glutathione-sepharose 4B affinity column pre-equilibrated with 1X PBS . The column was washed with 4 bed volumes of PBS to remove the unbound proteins. The bound fusion protein (GST-BNP (1-32)) was eluted with 5mM reduced glutathione in 50mM Tris/HCl, pH 8.0.The fusion protein was obtained was Approximately 3.0-3.1g of GST-BNP fusion protein

Cleavage of fusion protein:
The pure fractions of fusion protein were pooled and dialyzed into 20mM Tris/HCl, pH 7.5, 100mM NaCl and 1mM CaCl2, (FXa buffer) to remove the reduced glutathione. The dialyzed fused GST-hBNP protein was cleaved with FXa (1:200 w/w enzyme to fusion protein ratio) at 220 C for 2h. The cleavage of fusion protein was 85%. To collect the hBNP (1-32), the digested sample was reloaded onto the GST affinity column and the unbound protein was collected.

Reverse phase chromatography purification of Peptide:
The unbound fraction was further fractionated on Resource30 RPC to separate pure peptide from other contaminants like residual GST and host proteins. This fractionation resulted in purity greater than 99.0% when analyzed by analytical HPLC and the final yield of pure peptide was approximately 15-20mg/liter culture.

Table 1: Comparison on the purity and yield of hBNP produced using the GH fusion partner with that of GST fusion partner.

ADVANTAGES OF THE INVNETION:
According to the present invention, a large amount of desired peptide can be produced at low cost. Especially according to the present invention, the recovery process is minimized to a single step using a mild detergent without using any chaotropic agents.

We Claim:

1. A process for the production and recovery of the recombinant human BNP comprising the steps of:
a) transformation of host cells with an expression vector comprising a gene coding for fusion protein,
b) culturing the transformed host cell,
c) disruption of cells, separation and recovery of inclusion bodies,
d) solubilization of inclusion bodies with an anionic detergent and;
e) cleavage of the inclusion bodies and recovering rhBNP.
2. A process as claimed in claim 1, wherein the expression vector encoding the fusion protein comprises:
a) a DNA sequence encoding N-terminal portion of amino acid of truncated growth hormone as a fusion partner,
b) a linker encoding Ile-Glu-Gly-Arg,
c) a DNA sequence encoding the hBNP,
d) a regulatory sequence capable of directing expression of a fusion protein in a host cell.
3. A process as claimed in claim 2, wherein DNA sequence encoding N-terminal portion of amino acid of truncated growth hormone is modified to replace cysteine at postion 54 with serine, lysine or proline.
4. A process as claimed in claim 1, wherein the arginine of the linker sequence is bound either to the C terminus of the fusion partner or N-terminus of the peptide encoding region.
5. A process as claimed in claim 1, wherein the anionic detergent is sodium dodecyl sulphate in the range 0.1-1 %.

Dated this 9th day of April 2010

Omana Ramakrishnan

Of K & S Partners
Agent for the Applicant