DESC:TECHNICAL FIELD
The present disclosure relates to the field of biotechnology and protein science. Broadly, it describes a process for production of recombinant and stable proteases such as but not limited to Trypsin, Carboxypeptidase B and Kexin. Specifically, the present disclosure relates to a process for production of proteases in optimal quantities when expressed with a stable pro-peptide sequence. More particularly, the present disclosure relates to expression of these proteases with a stable pro-peptide sequence of Carboxypeptidase B, to produce a fusion protein. Also, the present disclosure relates to a process employing proteases to convert Insulin, its precursors and analogues to active proteins.

BACKGROUND AND PRIOR ART OF THE DISCLOSURE
Trypsin is a serine protease of ~24 kDa, found in the digestive system of many vertebrates, where it hydrolyses proteins. Trypsin is produced in the pancreas as the inactive protease trypsinogen. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids Lysine or Arginine. It is used for numerous biotechnological processes. In 1876, Trypsin was first named by Kuhne who described the proteolytic activity of this pancreatic enzyme. He compared Trypsin and Pepsin, discovering the differentiating factor to be the optimal pH. Today, Trypsin continues to be used in the development of cell and tissue culture protocols (Soleimani et al. 2009, Banumathi et al. 2009, and Yang et al. 2009).

Bovine pancreas expresses two forms of Trypsin, the dominant cationic and the minor anionic forms. These protein sequences share 72% identity, while their coding regions share 78% identity. Each of these proteins is further processed into alternate forms. Catalytic Trypsin contains a flexible “autolysis loop” (residues G145-V157) (Schroeder and Shaw 1968, and Bartunik et al. 1989), and autolysis of the dominant, single-chain form B-Trypsin at K148-S149 within this loop leads to the formation of A-Trypsin. Further autolysis at K193-D194 leads to the formation of Psi-Trypsin (Fehlhammer and Bode 1975).

Carboxypeptidase B is a metallocarboxypeptidase of weight ~34kDa, that catalyzes the hydrolysis of the basic amino acids Lysine, Arginine, and Ornithine from the C-terminal position of polypeptides. Carboxypeptidase B is a metalloenzyme, which is widely used for commercial and research purposes. Commercially available CPB purified from porcine or bovine pancreas is very expensive, and is not totally free from other proteases. Carboxypeptidases are secreted as zymogens by the acinar cells of the pancreas. The zymogens are activated by Trypsin in the small intestine. Procarboxypeptidase B is activated by Trypsin to form mature Carboxypeptidase B; which is highly specific for Lysine and Arginine, but shows preference for Arginine (Tan and Eaton 1995). It can also act (at a slower rate) on Valine, Leucine, Isoleucine, Asparagine, Glycine, and Glutamine (Villegas et al. 1995, Nishihira et al. 1995).

Kexin or Yeast Kex2 protease is a ~90kDa pro hormone processing endoprotease. Kex2 enzyme cleaves the a – factor precursor at the carboxyl side of Lys – Arg / Arg – Arg sites. Kex2 in the yeast Saccharomyces cerevisiae is a transmembrane, Ca2+-dependent serine protease of the Subtilisin-like pro-protein convertase (SPC) family with specificity for cleavage after paired basic amino acids. At steady state, Kex2 is predominantly localized in late Golgi compartments and initiates the proteolytic maturation of pro-protein precursors that transit the distal secretory pathway. The Kex2 protein sequence suggests several post– translational modifications. The NH2 – terminus contains a probable signal peptide (residues 1- 19), and a single hydrophobic transmembrane domain (residues 679 – 699) and a 115 residue cytosolic tail.

The present disclosure overcomes the drawbacks of the methods of the prior art by a pro-peptide sequence for fusion to recombinant proteases, fusion proteins of pro-peptide sequence and proteases, a process for production of recombinant proteases employing the pro-peptide, and a process employing proteases for activation of Insulin, its precursors and analogues.

STATEMENT OF THE DISCLOSURE
Accordingly, the present disclosure relates to a fusion protein comprising propeptide sequence of Carboxypeptidase B, and a Protease; a recombinant nucleic acid molecule encoding the fusion protein as above; a vector comprising the recombinant nucleic acid molecule as above; a host cell comprising the vector as above; a process for producing a recombinant protease, said process comprising steps of – a) Introducing a vector which comprises recombinant nucleic acid molecule as above and encoding the fusion protein as above, into a host cell, b) Culturing the host cell to produce the fusion protein, and c) Cleaving the fusion protein to produce the recombinant protease; and a process of converting protein precursor selected from the group comprising Insulin, Glargine, Aspart, Lispro, Glulisine, Insulin Detemir and combinations thereof, into active protein, said process comprising act of contacting the protein precursor with protease selected from the group comprising Carboxypeptidase B, Trypsin, Kexin, Enterokinase, Asp N endoproteinase and Lysine endopeptidase.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:

Figure 1 depicts pADB3 vector map.

Figure 2 depicts pADB3 – ProCPB - Carboxypeptidase B vector map.

Figure 3 depicts pADB3 – ProCPB - Trypsinvector map.

Figure 4 depicts pADB3 – ProCPB- Kexin vector map.

Figure 5 depicts analysis of different clones of ProCPB- Carboxypeptidase on SDS PAGE.
• Lane M: blue pre-stained ladder;
• Lane GS115: Host control induced with Methanol;
• Lane 1, 2, 3, 4, 5, 6: Clones 1 – 6 induced with Methanol.

Figure 6 depicts analysis of different clones of ProCPB -Trypsin on SDS PAGE.
• Lane M: blue pre-stained ladder;
• Lane GS115: Host control induced with Methanol;
• Lane NPMe: Host control without Methanol;
• Lanes 1, 2, 3, 4, 5, 6 and 7: Clones 1 – 7 induced with Methanol.
Figure 7 depicts analysis of different clones of ProCPB- Kexin on SDS PAGE.
• Lane GS115: Host control induced with Methanol;
• Lane M: blue pre-stained ladder;
• Lanes 1, 2, 3, 4, 5, 6, 7 and 8: Clones 1 – 8 induced with Methanol.

Figure 8 depicts Reverse Phase HPLC profile of Carboxypeptidase B.

Figure 9 depicts Reverse Phase HPLC profile of Trypsin.

Figure 10 depicts SDS PAGE image of Carboxypeptidase B.

Figure 11 depicts SDS PAGE image of Trypsin.

Figure 12 depicts Reverse Phase HPLC profile of Kexin.

Figure 13 depicts Overlay Chromatogram of proCPB-Trypsin treated and untreated tagged Insulin protein.

Figure 14 depicts Overlay chromatogram of activated Trypsin treated and untreated tagged Insulin protein.

Figure 15 depicts Overlay chromatogram of proCPB-Carboxypeptidase treated and untreated tagged Insulin protein.

Figure 16 depicts Overlay chromatogram of activated Carboxypeptidase B treated and untreated tagged Insulin protein.

Figure 17 depicts Overlay chromatogram of proCPB-Kexin treated and untreated tagged Insulin protein.

Figure 18 depicts Overlay chromatogram of activated Kexin treated and untreated tagged Insulin protein.

Figure 19 depicts purity of Kexin by SDS PAGE.
Lane 1: molecular weight marker;
Lane 2 to lane 4: different fractions of purified Kexin from Ion Exchange Chromatography.

Figure 20 depicts analysis of different clones of Kexin and Pro CPB Kexin on SDS PAGE.
• Lane GS115: Host control induced with Methanol;
• Lane M: blue pre-stained ladder;
• Lanes 1 and 2: Kexin and Pro CPB clones 1 and 2 induced with Methanol

DETAILED DESCRIPTION OF THE DISCLOSURE
The present disclosure relates to a fusion protein comprising propeptide sequence of Carboxypeptidase B; and a Protease.

In an embodiment of the present disclosure, the protease is selected from the group comprising Enterokinase, Lysine endopeptidase, Asp N endoproteinase Carboxypeptidase B, Trypsin and Kexin.

In another embodiment of the present disclosure, the fusion protein comprises amino acid sequence selected from the group comprising SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7.

The present disclosure also relates to a recombinant nucleic acid molecule encoding the fusion protein as above.

In an embodiment of the present disclosure, the nucleic acid molecule comprises pro-peptide sequence of SEQ ID NO: 1.

The present disclosure also relates to a vector comprising the recombinant nucleic acid molecule as above.

The present disclosure also relates to a host cell comprising the vector as above.

In an embodiment of the present disclosure, the host cell is selected from the group comprising – E.coli, Saccharomyces cerevisiae, Hansenula, Bacillus, Yarrowia Kluveromyces, and Pichia pastoris.
The present disclosure also relates to a process for producing a recombinant protease, said process comprising steps of:
a) Introducing a vector which comprises recombinant nucleic acid molecule as above and encoding the fusion protein as above, into a host cell;
b) Culturing the host cell to produce the fusion protein; and
c) Cleaving the fusion protein to produce the recombinant protease.

In an embodiment of the present disclosure, the fusion protein is cleaved by method selected from the group comprising autocatalytic cleavage, change in pH, increased temperature, active enzymes and combinations thereof.

The present disclosure also relates to a process of converting protein precursor selected from the group comprising Insulin, Glargine, Aspart, Lispro, Glulisine, Insulin Detemir and combinations thereof, into active protein, said process comprising act of contacting the protein precursor with protease selected from the group comprising Carboxypeptidase B, Trypsin, Kexin, Enterokinase, Asp N endoproteinase, Lysine endopeptidase.

In an embodiment of the present disclosure, the protease is the recombinant protease produced by the process as above.

The present disclosure relates to enhanced and high density cell expression of recombinant protease, with the help of a pro-peptide sequence.

In an embodiment, a pro-peptide sequence is attached or fused or combined to the protease as an N-terminal tag protein, to give a fusion protein.

The present disclosure also relates to a Fusion protein of protease and pro-peptide.

In embodiments of the present disclosure, the protease is selected from the group comprising Carboxypeptidase B, Trypsin, Kexin, Enterokinase, Asp N endoproteinase, Lysine endopeptidase.
In an embodiment of the present disclosure, the protease is Trypsin.
In an embodiment of the present disclosure, the protease is Carboxypeptidase B.

In an embodiment of the present disclosure, the protease is Kexin.

Enzymes are synthesized as inactive precursors (Zymogens) in vivo. Under appropriate conditions, Zymogens are activated to form the mature active protein. For activation, there is cleavage of an amino-terminal peptide which is referred to as the "pro-peptide" sequence.

In an embodiment of the present disclosure, the pro-peptide is pro-peptide region of Bovine Carboxypeptidase B (94 amino acids). It is also referred to as “Pro-region" or "Pro-sequence" or
“ProCPB” or “Pro-CPB” throughout the present disclosure. It is also referred to as “tag”, “tag protein” and “N-terminal tag protein” throughout the disclosure.

In an embodiment, the pro-peptide sequence is represented by SEQ ID NO: 1 of the present disclosure.

In an embodiment of the present disclosure, the pro-peptide is a sequence having about 85% to 95% homology to pro-peptide sequence of Bovine Carboxypeptidase B.

In an embodiment of the present disclosure, the pro-peptide is a sequence having about 85% to 95% homology to SEQ ID NO: 1.

In embodiments of the present disclosure, “untagged protein” is defined as a protein or specifically, a protease, to which, the pro-peptide sequence or tag of the present disclosure was not attached.

In an embodiment of the present disclosure, the attachment or combination or fusion of pro-peptide and protease is termed as a Fusion protein.

In an embodiment, the Fusion protein is ProCPB – Carboxypeptidase B.

In an embodiment, the Fusion protein ProCPB – Carboxypeptidase B is represented by SEQ ID NO: 3.

In an embodiment, the Fusion protein is ProCPB –Trypsin.

In an embodiment, the Fusion protein ProCPB –Trypsin is represented by SEQ ID NO: 5.

In an embodiment, the Fusion protein is ProCPB –Kexin.

In an embodiment, the Fusion protein ProCPB –Trypsin is represented by SEQ ID NO: 7.

The present disclosure also relates to recombinant nucleic acid molecule encoding the fusion protein of the present disclosure.

The recombinant nucleic acid molecule comprises nucleotide sequence of pro-peptide sequence and nucleotide sequence of protease.

In an embodiment, the recombinant nucleic acid molecule comprises nucleotide sequence of pro-peptide sequence of Bovine Carboxypeptidase B, represented by SEQ ID NO: 1.

In an embodiment, the recombinant nucleic acid molecule also comprises components needed for expression of the fusion protein, including but not limited to components such as promoter, transcription terminator sequence, leader sequence, polyadenylation sequence, signal peptide coding sequence, etc.

The nucleic acid molecule encoding the fusion protein may comprise additional components apart from the nucleic acid sequence of pro-peptide and nucleic acid sequence of protease. Additional components include but are not limited to nucleic acid sequences encoding signal peptide, mat alpha signal sequence, HSA signal sequence, amylase signal sequence.
In an embodiment of the present disclosure, the Fusion protein undergoes cleavage for removal of the pro-peptide sequence and providing the recombinant protease.

In an embodiment, the recombinant fusion protease is recovered intact from the culture medium.

In an embodiment, upon secretion of the fusion protein from the host cell, the pro-peptide sequence is cleaved after initial purification, and the active recombinant protease obtained is used for cleavage of insulin, its precursors and analogues.

In embodiments of the present disclosure, the recombinant protease is expressed in a variety of host cells, for e.g., but not limiting to bacterial system such as E.coli as well as fungal cells such as Pichia, Saccharomyces, Yarrowia, Candida and Hansenula.

In an embodiment of the present disclosure, the nucleotide sequence encoding enzyme Trypsin is codon-optimized.

In an embodiment of the present disclosure, the nucleotide sequence encoding enzyme Carboxypeptidase B is codon-optimized.

In an embodiment of the present disclosure, the nucleotide sequence encoding enzyme Kexin is codon-optimized.

In an embodiment of the present disclosure, the nucleotide sequence encoding pro-peptide sequence of Bovine Carboxypeptidase B is codon-optimized.

In embodiments of the present disclosure, codon optimization of the protease sequence, in combination with the specific pro-peptide sequence provides enhanced expression of the protease and is one of the important features of the present disclosure.

The expression system described in the present disclosure includes the usage of novel pro-peptide sequence as tag protein for recombinant protein expression and secretion of recombinant Fusion protein into the culture supernatant.

The present disclosure further relates to cloning the polynucleotides encoding such fusion proteins, and to expression vectors for expression of such fusion proteins. These expression vectors are further transformed into host cells to generate recombinant, stable and inactive fusion protein.

The present disclosure also relates to usage of the above mentioned recombinant Proteases in order to convert Insulin precursors to the active protein.

The use of pro-peptides in the present disclosure involves the use of pro-peptides native to the host and fusion of the same to the mature protein or protein with appropriate signal peptides for efficient translocation to specific compartments in the host or to the extracellular culture medium as demonstrated by SDS PAGE results of the present disclosure.

In an embodiment, the present disclosure also relates to a method for for activating inactive proteases by removal of tag, i.e., pro-peptide, in turn activating the protease.

The culture medium, reagents and culture conditions employed in the processes of the present disclosure are known from standard culture techniques and information available. A person skilled in the art, upon reading the present disclosure, would be able to determine the appropriate culture medium, reagents and culture conditions for the processes of the present disclosure.

In an embodiment, the present disclosure also relates to a high cell density fermentation process of Trypsin, Carboxypeptidase or Kexin.

The process after fermentation includes:
1. Purification of protease by affinity chromatography.
2. Purification of protease by ion exchange chromatography.
3. Thus prepared Protease such as Trypsin, Carboxypeptidase B or Kexin is used in process development of Insulin, Glargine Lispro, Aspart and all insulin analogues.

Being a potent protease, Trypsin cannot be produced in its catalytically active form. Both the cationic and anionic trypsin proteins are expressed as trypsinogen proenzymes, with a 15-residue signal peptide (M1-A15) and an 8-residue pro-peptide (F16-K23). In addition, the catalytic triad and regions flanking the catalytic triad are highly conserved (Hartley 1970). Hence it is a very big challenge to express Trypsin in its active form. Moreover, the expression level is very low. To overcome this challenge, in an embodiment of the present disclosure, a a pro-peptide sequence of 94 amino acids, i.e. the pro-peptide of Bovine Carboxypeptidase B, is expressed along with Bovine cationic Trypsinogen.

Also, when Carboxypeptidase is expressed in Pichia in its active form, it gets degraded in the culture supernatant.

To overcome this challenge, in an embodiment of the present disclosure, its own pro-peptide sequence is used as a tag protein, which is of 94 amino acids length.

Further, amino acid sequence analysis revealed that the N terminus of mature Kex2 protease is created by a potentially auto proteolytic cleavage at Lys108 – Arg109, prior to the domain homologous to Subtilisin, followed by trimming of Leu – Pro and Val – Pro dipeptides by Ste13 dipeptidyl aminopeptidase. Moreover it is a 90 kDa protein, known for autocatalytic activity. Hence a tag which is of 94 amino acids, i.e. Pro-peptide sequence of Bovine Carboxypeptidase B, is used for expression of Kex2 protease.

In an embodiment of the present disclosure, codon optimized trypsin gene of ~ 690bp is linked to ~282bp pro-peptide of Bovine Carboxypeptidase B gene cloned into pADB3 vector, under the control of AOX1 promoter and secretion signal Mat alpha. The resulting plasmid is introduced into Pichia pastoris by standard transformation techniques. Several clones are evaluated by inducing with Methanol by standard protocol.

In embodiments of the present disclosure, Carboxypeptidase and Kexin genes are similarly cloned into pADB3 vectors and several clones are evaluated for stable protein expression.

The present disclosure envisages the use of a pro-peptide sequence which protects the protease from its autocatalytic activity. It further reduces protease activation and degradation due to change in the media pH. Proteases expressed with the tag protein remain inactive until it is activated by external means. The present disclosure also provides the production of proteases in higher quantities, compared to untagged proteins, by using expression vector.
The present disclosure overcomes the drawbacks of the methods of the prior art by increasing the yield, stability and storage time of protease enzymes. Usage of ProCPB region of ~10kDa provides for efficient expression of inactive Trypsin or Carboxypeptidase or Kexin, which are known for their autocatalytic activity, when in their mature form.
The present disclosure is further described with help of the following examples. However, these examples should not be construed to limit the scope of the present disclosure.

EXAMPLES
EXAMPLE 1: CLONING AND EXPRESSION OF PRO CPB - CARBOXYPEPTIDASE B
The utility of the present disclosure is demonstrated by construction of expression plasmids wherein codon optimized gene sequence of Pro-peptide region of Bovine Carboxypeptidase B (Hereinafter referred to as Pro-CPB) represented by SEQ ID NO: 1 is fused to Carboxypeptidase B gene (SEQ ID NO: 2) and inserted into expression vector pADB3 and expressed in Pichia pastoris GS115 strain.

1. Isolation of BstBI and EcoRI vector backbone of pADB3:
About 5.8 µg of pADB3 plasmid (Anthem’s in house vector) is suspended in Cutsmart buffer (NEB). To this, 3µl of EcoRI and 3µl of XhoI restriction enzyme is added and incubated at 37°C for 2 hours. After 2 hours of incubation, the reaction mixture is electrophoresed on 1% agarose gel at 120V. Vector backbone of ~5944bp is excised and gel eluted.This is used for ligation.

2. Synthesis of Pro Carboxypeptidase gene:
The coding region of Pro-peptide of Bovine Carboxypeptidase gene (SEQ ID NO: 1) and Carboxypeptidase gene (SEQ ID NO: 2), after codon optimization using codons commonly found and highly expressed in Yeast, is designed to include convenient restriction sites for cloning into pADB3 vector.

Figure 1 of the present disclosure provides the vector map and Table 1 provides the vector components for pADB3 vector.

Table 1
Component Nucleotides
AOX 1 Promoter 18 – 942
Mat Alpha Signal sequence 957 – 1199
MCS 1200 - 1259
AOX1 Terminator 1260 - 1587
Kanamycin 1980 - 2795
3’AOX1 3255 – 3998
Ampicillin 4360 - 5220
pUC origin 5298 – 5940

3. Isolation of Pro-peptide region of Bovine Carboxypeptidase gene:
About 2.6µg of plasmid Bovine Pro Carboxypeptidase / pMA is suspended in Cutsmart buffer (NEB) To this, 5µl of EcoRI and 5µl of XhoI restriction enzyme is added and incubated at 37°C for 2 hours. After 2 hours of incubation, the reaction mixture is electrophoresed on 1% agarose gel at 120V. Insert of ~1266 bp is excised and gel eluted. This is used for ligation.

4. Ligation, Transformation and Screening :
pADB3 digested with XhoI and EcoRI restriction enzyme yielded ~5944bp vector band. Bovine Pro Carboxypeptidase digested with XhoI and EcoRI restriction enzyme gave ~ 1266bp insert band. Based on the concentration of vector and insert, ligation reaction is set in the ratio 1:3 and 1:4 and incubated overnight at 16ºC + 0.5°C. Next day, this ligation mixture is transformed into E.coli DH5a competent cells. Transformation mix is plated onto LB Amp (100µg/ml) plates. These plates are incubated at 37°C for overnight growth. Eighteen clones are screened by colony PCR using pPIC9KFP (SEQ ID NO: 8) and pPIC9KRP (SEQ ID NO:9) primers. All Eighteen clones are found to be positive, of which clone 4 is used for further analysis.

5. Transformation into Pichia pastoris and screening for multicopy integrants:
Pro Carboxypeptidase / pADB3 # 4 vector, as depicted in figure 2 of the present disclosure, is linearized using SacI and transformed into electro competent cells of Pichia Pastoris GS115 by electroporation. Protocol is followed as described in Invitrogen manual. Regeneration mix is isolated onto YPD (Yeast Extract Peptone Dextrose) with 100µg/ml G418 plates. From the transformed plates, approximately 300 colonies are inoculated in YPD broth in 96 well micro titer plates along with appropriate controls. The plates are incubated at 30°C for 24 hours and then replica plated onto YPD agar plates containing 1mg/ml G418. Six colonies resistant for 1mg/ml G418 are selected and induced for Pro Carboxypeptidase expression.

6. Small scale expression studies:
A small scale expression study is carried out in shake flasks. Briefly, the clones are grown at 30°C in BMGY (Buffered Glycerol Complex Medium), followed by induction with Methanol in BMMY (Buffered Methanol-Complex Medium) at 30°C. Induction with Methanol is carried out for a total of 3 days. Eight clones are taken for expression studies. Further induction is carried out for a total of 4 days. It is observed from Figure 5 of the present disclosure that compared to host control i.e., GS115 host (without any plasmid insertion) when induced with Methanol similar to that of other clones, shows no other protein bands on the SDS PAGE corresponding to the molecular weight of Pro Carboxypeptidase. and usage of ProCPB fusion protein has increased the stability and yield of the Carboxypeptidase enzyme thus retaining it in its inactive form. .

EXAMPLE 2: CLONING AND EXPRESSION OF PROCPB -TRYPSIN
The utility of the present disclosure is demonstrated by construction of expression plasmids wherein codon optimized gene sequence of Pro CPB (SEQ ID NO: 1) is fused to Trypsin gene (SEQ ID NO: 4) and inserted into expression vector pADB3 and expressed in Pichia pastoris.

1. Isolation of BstBI and EcoRI vector backbone of pADB3:
About 600ng of ProCPB/pADB3 plasmid (Anthem’s in house vector) is suspended in Cutsmart buffer (NEB). To this, 3µl of EcoRI restriction enzyme is added and incubated at 37°C for 2 hours. After 2 hours of incubation, 3µl of BstBI enzyme is added and incubated at 65°C for 2 hours. Reaction mixture is electrophoresed on 1% agarose gel at 120V. Vector backbone of 5687bp (from which ~1475bp of ProCPB gene with Mat-alpha signal sequence signal sequence is removed) is excised and gel eluted.This is used for ligation.

2. Synthesis of ProCPB -Trypsin gene:
The coding region of Pro-peptide sequence of Bovine CPB gene (SEQ ID NO: 1) and Trypsinogen gene (SEQ ID NO: 4), after codon optimization using codons commonly found in highly expressed Yeast, is fused by overlapping PCR (SEQ ID NO: 05) and designed to include convenient restriction sites for cloning into pADB3 vector.

3. Isolation of ProCPB -Trypsin gene:
About 2.5µg of plasmid ProCPB -Trypsin is suspended in Cutsmart buffer (NEB). To this, 3µl of EcoRI restriction enzyme is added and incubated at 37°C for 2 hours. After 2 hours of incubation, 3µl of BstBI enzyme is added and incubated at 65°C for 2 hours. Reaction mixture is electrophoresed on 1% agarose gel at 120V. Insert of 990bp is excised and gel eluted. This is used for ligation.

4. Ligation transformation and screening:
pADB3 digested with XhoI and EcoRI restriction enzyme yielded 5687bp vector band. ProCPB Trypsindigested with XhoI and EcoRI restriction enzyme gave 990bp insert band. Based on the concentration of vector and insert, ligation reaction is set in the ratio 1:3 and 1:4 and incubated overnight at 16ºC + 0.5°C. Next day, this ligation mixture is transformed into E.coli DH5a competent cells. Transformation mix is plated onto LB Amp (100µg/ml) plates. These plates are incubated at 37°C for overnight growth. Twelve clones are screened by restriction digestion using BstBI and EcoRI restriction enzymes. Of all twelve colonies; clone 5 is used for further analysis.

5. Transformation into Pichia pastoris and screening for multicopy integrants:
ProCPB-Trypsin/ pADB3 # 5 vector, as depicted in figure 3 of the present disclosure, is linearized using SacI and transformed into electro competent cells of Pichia pastoris GS115 by electroporation. The protocol is followed as described in Invitrogen manual. Regeneration mix is plated onto YPD with 100µg/ml G418 plates. From the transformed plates, approximately 400 colonies are inoculated in YPD broth in 96 well microtitre plates along with appropriate controls. The plates are incubated at 30°C for 24 hours and then replica plated onto YPD agar plates containing 1mg/ml G418. Eight colonies resistant for 1mg/ml G418 are selected and induced for ProCPB Trypsin expression.
6. Small scale expression studies:
A small scale expression study is carried out in shake flasks. Briefly, the clones are grown at 30°C in BMGY, followed by induction with Methanol in BMMY at 30°C. Induction with Methanol is carried out for a total of 3 days. Eight clones are taken for expression studies. Further induction is carried out for a total of 4 days. It is observed from

It is observed from Figure 6 of the present disclosure that compared to host control i.e., GS115 host (without any plasmid insertion) when induced with Methanol similar to that of other clones, shows no other protein bands on the SDS PAGE corresponding to the molecular weight of ProCPB Trypsinogen. And usage of ProCPB fusion protein has increased the stability and yield of the ProCPB Trypsin enzyme, thus retaining it in its inactive form.

EXAMPLE 3: CLONING AND EXPRESSION OF PRO CPB - KEXIN
The utility of the present disclosure is demonstrated by construction of expression plasmids wherein codon optimized gene sequence of Pro CPB (Seq ID NO: 1) is fused to Kexin gene (SEQ ID NO: 6) and inserted into expression vector pADB3 and expressed in Pichia pastoris.
1. Isolation of XhoI and EcoRI vector backbone of pADB3:
About 4.6µg of pADB3 plasmid (Anthem’s in house vector) is suspended in Cutsmart buffer (NEB). To this, 5µl of EcoRI and 5µl of XhoI restriction enzyme is added and incubated at 37°C for 2 hours. After 2 hours of incubation, the reaction mixture is electrophoresed on 1% agarose gel at 120V. Vector backbone of ~5944bp is excised and gel eluted.This is used for ligation.

2. Synthesis of PCPB Kexin gene:
The coding region of Pro-peptide sequence of Bovine CPB gene (SEQ ID NO: 1) and Kexin gene (SEQ ID NO: 6), after codon optimization using codons commonly found in highly expressed Yeast, is fused by overlapping PCR (SEQ ID NO: 07) and designed to include convenient restriction sites for cloning into pADB3 vector, as shown in Figure 1.

3. Isolation of ProCPB -Kexin gene:
About 4.6µg of plasmid Pro-CPB- Kexin is suspended in Cutsmart buffer (NEB). To this, 5µl of EcoRI and 5µl of XhoI restriction enzyme is added and incubated at 37°C for 2 hours. After 2 hours of incubation, the reaction mixture is electrophoresed on 1% agarose gel at 120V. Insert of ~1983bp is excised and gel eluted. This is used for ligation.

4. Ligation transformation and screening:
pADB3 digested with XhoI and EcoRI restriction enzyme yielded 5944bp vector band. Pro-CPB Kexin digested with XhoI and EcoRI restriction enzyme gave 1983bp insert band. Based on the concentration of vector and insert, ligation reaction is set in the ratio 1:1 and incubated overnight at 16ºC + 0.5°C. Next day, this ligation mixture is transformed into E.coli DH5a competent cells. Transformation mix is plated onto LB Amp (100µg/ml) plates. These plates are incubated at 37°C for overnight growth. Twelve clones are screened by colony PCR, of which clone 5 is used for further analysis.

5. Transformation into Pichia pastoris and screening for multicopy integrants:
ProCPB- Kexin/ pADB3 # 5 vector, as depicted in figure 4 of the present disclosure, is linearized using SacI and transformed into electro competent cells of Pichia pastoris GS115 by electroporation. Protocol is followed as described in Invitrogen manual. Regeneration mix is plated onto YPD with 100µg/ml G418 plates. From the transformed plates, approximately 300 colonies are inoculated in YPD broth in 96 well microtitre plates along with appropriate controls. The plates are incubated at 30°C for 24 hours and then replica plated onto YPD agar plates containing 1mg/ml G418. Ten colonies resistant for 1mg/ml G418 are selected and induced for ProCPB -Kexin expression.

6. Small scale expression studies:
A small scale expression study is carried out in shake flasks. Briefly, the clones are grown at 30°C in BMGY, followed by induction with Methanol in BMMY at 30°C. Induction with Methanol is carried out for a total of 3 days. Eight clones are taken for expression studies. Further induction is carried out for a total of 4 days.

It is observed from Figure 7 and Figure 20 of the present disclosure that, compared to host control i.e., GS115 host (without any plasmid insertion) and Kexin clones when induced with Methanol similar to that of other clones, shows no other protein bands on the SDS PAGE corresponding to the molecular weight of ProCPB Kexin / Kexin; And usage of ProCPB fusion protein has increased the stability and yield of the ProCPB Kexin enzyme, thus retaining it in its inactive form.

As a result of Examples 1-3, activity of enzyme is found to be 1195 units/ml for Trypsin, 1345 units/ml for Carboxypeptidase B and 1213 units/ml for Kexin. Thus, it is observed that the use of a pro-peptide sequence in a fusion protein with protease, is highly useful for the stability, yield and activity of proteases.

EXAMPLE 4: FERMENTATION PROCESS FOR THE PRODUCTION OF PROTEASE
The recombinant Pichia pastoris fermentation process is developed with fed-batch and continuous method. Initial glycerol stock is inoculated to YPD media (table 2) and incubated for 20-30 hours in incubatory shaker at 30°C and 250 rpm. The growth is monitored by OD600 (optical density at 600nm) in a spectrophotometer and transferred to pre-production flask, consisting of Glycerol Salt medium (table 3) between 15-20 OD600 value. Similarly, the pre-production flask is incubated for 24 -30hrs and transferred to Fermenter between the OD 600 values of 12-15.

Initial fermentation media consists of Glycerol salt media supplemented with additional trace salts such as Copper Sulphate (5-8g/L), Manganese Sulphate (1-5g/L), Ferrous Sulphate (60-70g/L), Magnesium Sulphate (10-20g/L), Zinc Chloride (15-25g/L), Cobalt Chloride (0.5-2g/L), Boric Acid (0.01-0.05g/L), Sodium Molybdate (0.1-0.5g/L), Sodium Iodide (0.05-0.01g/L) and D-Biotin (0.2-0.5g/L). The individual salts are dissolved in minimal quantity of water and sterilized for 60 minutes at 121°C. The trace salts solution and D-Biotin is added aseptically after filter sterilization, to the fermentation medium.

The fermentation process consists of batch phase (growth phase), an additional Glycerol fed batch phase (exponential growth phase) and Methanol fed batch phase (induction phase).

Batch Phase
Batch phase fermenter monitoring and control are mentioned below:

Initial Fermenter parameters
Temperature : 30 ± 2°C
RPM : 300 rotations per minute
pH : 5.0±0.2
DO (dissolved oxygen): > 50%
Airflow : 0.5 LPM/L of broth.

During the fermentation, the dissolved oxygen is maintained by altering the process parameters such as RPM, airflow, back pressure and by supplying oxygen.

Glycerol fed batch phase
The Glycerol fed batch phase starts when the batch phase ends between 24-30 hours. Glycerol feed rates are gradually increased from 2g/L/h to 50g/L/h depending upon the accumulation. During the exponential feeding phase, the pH is maintained between 5.0 ± 0.2 by using 50% Orthophosphoric acid and 50% Ammonia solution.

Methanol Induction phase
Methanol feeding starts immediately at the end of Glycerol fed batch phase, once the packed cell volume reaches about 300-400g/L and is also monitored by increase in dissolved oxygen values and pH. During the Methanol induction phase, temperature is reduced to 24±2°C and pH is maintained between 3.0-5.0, more specifically between 3.5-4.0. Methanol feed rate varies between 2g/L/h to 40g/L/h during the feed phase and depends upon the uptake and accumulation values. Similarly, Nitrogen feed (table 4) is also provided at the rate of 1-5g/L/h to the culture.

Table 2 - YPD media
Components Concentration (g/L)
Yeast extract 10
Peptone 20
Dextrose 20

Table 3- Glycerol salt media
Components Concentration (g/L)
CaSO4 1
K2SO4 30
MgSO4 25
Orthophosphoric Acid 27
Glycerol 50
KOH 5

Table 4- Nitrogen feed
Components Concentration (g/L)
Soya flour Hydrolysate 100
Yeast Extract 40
Soya Peptone 80

The Batch is harvested when the product formation reduces, after about 10-15days.

EXAMPLE 5: PRIMARY DOWNSTREAM PROCESSING
At the end of fermentation, broth and partial harvest are centrifuged at 4-8°C for 40 minutes at 4500 rpm. The supernatant is collected and further processed by Microfiltration, dia-filtration and ultrafiltration. . Microfiltration and ultrafiltration is carried out at 2-15°C at 1 TMP (trans membrane pressure) at a flux of 10L/h. After microfiltration, buffer exchange carried out using diafiltration technique and is carried out for 3-5 volumes. During ultrafiltration, the volume is reduced to 10 fold. Microfiltration is carried out with 0.1/0.2 micron pore size and ultrafiltration is carried out with a 4 kDa cut off membrane.

EXAMPLE 6: ENZYME ACTIVATION
Micro filtrated and ultra-filtered pro-Trypsin is activated by adding TRIS 50g/L and CaCl2 4g/L and 100 IU/ml of sample at pH 8.5. The activation of the enzyme is triggered by removal of the tag, i.e. the pro-peptide sequence.

The reaction mixture is incubated at a temperature between 4-25°C. The conversion is monitored and after 4-15 hours, the reaction is arrested by bringing down the pH of the solution to 2.5 and stored at -20?C for further processing.

EXAMPLE 7: AFFINITY CHROMATOGRAPHY
The column is packed with benzamine Sepharose – FF and is equilibrated with equilibration buffer having 10 to 100 mM mM, more preferably 40-60mM , 100 mM to 1000 mM, more preferably 400-600mM Sodium Chloride, at pH-3.5-9.5 preferably 6.5-7.5 . The activated Trypsin from Example 6 is loaded on to the column and eluted with elution buffer containing 10-250 mM glycine, preferably 100-200 mMat pH 2.0-4.5 in 15 to 35 column volume and further loaded on the ion exchange column

EXAMPLE 8: ION EXCHANGE CHROMATOGRAPHY
SP ImpRes is packed in a glass column and equilibrated with TRIS buffer at pH 5.5-8.5 and activated Trypsin from Example 6 is loaded on to the column. A post load of 5 Column volume is given and Trypsin is eluted with 10-200mM TRIS with 0.5-1.5M NaCl in a linear gradient of 10-35 CV. Purified Trypsin is collected,

The purity obtained at this step is more than 95% as determined by HPLC and SDS PAGE) of figures 8 and 11 respectively of the present disclosure.

EXAMPLE 9: ION EXCHANGE CHROMATOGRAPHY
CM Sepharose is packed in a glass column and equilibrated with TRIS buffer at pH 4.5-6.5 and activated Carboxypeptidase B is loaded on to the column. A post load of 5 Column volume is given and Carboxypeptidase B is eluted with 50-250mM TRIS with 0.5-1.5M NaCl in a linear gradient of 10-35 CV. Purified Carboxypeptidase B is collected whose purity is found to be more than 95% as determined by SDS PAGE and HPLC of figures 9 and 10 respectively of the present disclosure..

EXAMPLE 10: ION EXCHANGE CHROMATOGRAPHY
DEAE Sepharose is packed in a glass column and equilibrated with TRIS buffer at pH 4.5-8.5 and activated Kexin is loaded on to the column. A post load of 5-15 Column volume is given and Kexin is eluted with 50-250mM TRIS with 0.5-1.5M NaCl in a linear gradient of 5-20 CV. Purified Kexin is collected whose purity is found to be more than 95% as determined by SDS PAGE of figure 19 of the present disclosure..

EXAMPLE 11: CLEAVING INSULIN PRECURSORS
When the fusion protein with the pro-peptide sequence is used in the process for enzymatic conversion of Insulin and its precursors, the conversion does not happen whereas when the protease enzyme is used in the process after activation, the conversion is complete, which is monitored by HPLC. During enzymatic conversions alkaline pH (7.0-10.5) is maintained and Trypsin/Kexin (2 to 20 units per 1 mg of insulin, glargine and aspart) and CPB (0.2 to 1.5 units per 1 mg of ( insulin, lispro and aspart) enzymes are used. The reaction is carried out at temperature 2-30°C for 5-20 hours. CaCl2 at a concentration of 2 to 100 mM is added as a divalent ion during the reaction.

This indicates that the proteases such as Trypsin, Carboxypeptidase B and Kexin are expressed in inactive form using the tags or pro-peptide sequences (in Protrypsin, Procarboxypeptidase and Pro-Kexin form).

It is also observed from figures 13 to 18 that usage of these proteases converts pro-peptide to active peptide such as Insulin, Glargine, Lispro and Aspart.

SEQUENCE LISTING

<110> ANTHEM BIOSCIENCES PVT. LTD.

<120> FUSION PROTEIN AND PROCESSES THEREOF

<130> IP30038

<140> 756/CHE/2015
<141> 2015-08-16

<160> 9

<170> PatentIn version 3.5

<210> 1
<211> 279
<212> DNA
<213> Artificial Sequence

<220>
<223> Nucleotide sequence of Pro CPB gene

<220>
<221> gene
<222> (1)..(279)

<400> 1
cattctggtg aacacttcga gggtgacaag gttttcagag ttcacgttga ggacgagaac 60

cacatctctt tgttgcacga attggcttcc accagacaga tggatttctg gaagccagac 120

tccgttactc aggttaagcc acactctacc gttgacttca gagttaaggc tgaggacact 180

gttgctgtcg aggatttctt gggtcagaac ggtttgagat acgaggtctt gatctccaac 240

ctgagatcca tgttggaggc tcaattcgac tccagagtt 279

<210> 2
<211> 924
<212> DNA
<213> Artificial Sequence

<220>
<223> Nucleotide sequence of Carboxypeptidase gene

<220>
<221> gene
<222> (1)..(924)

<400> 2
actactggtc actcctacga gaagtacaac aactgggaga ctatcgaggc ttggactgaa 60

caagttgctt ctgagaaccc agacctgatt tccagatccg ctatcggtac taccttcctg 120

ggtaacacca tctacttgct gaaggttggt aagccaggtt ccaacaagcc agctgttttc 180

atggattgtg gtttccacgc tagagagtgg atttccccag ctttctgtca gtggttcgtt 240

agagaggctg ttagaaccta cggtagagaa atccacatga ccgagttctt ggacaagctg 300

gacttctacg ttttgccagt cgttaacatc gacggttaca tctacacttg gaccaccaac 360

agaatgtgga gaaagaccag atctactaga gccggttctt cctgtactgg tactgacctg 420

aacagaaact tcgacgctgg ttggtgttct atcggtgctt ctaacaaccc atgctccgag 480

acttactgtg gttctgctgc tgaatctgag aaagagtcca aggctgttgc cgacttcatc 540

agaaaccact tgtcctccat caaggcctac ttgactattc actcctactc ccagatgatg 600

ctgtacccat actcctacga ctacaagctg ccaaagaaca acgtcgagtt gaacactttg 660

gctaagggtg ccgttaagaa attggcttcc ttgcacggta ctacctacac ttatggtcca 720

ggtgcctcta ctatctaccc agcttctggt ggttctgatg attgggctta cgaccagggt 780

atcaagtact ccttcacctt cgagttgaga gacaagggta gatacggttt cgttttgcca 840

gagtcccaga tccagccaac ttgtgaagaa actatgctgg ctatcaagta cgtcacctcc 900

tacgttttgg agcacttgta ctaa 924

<210> 3
<211> 402
<212> PRT
<213> Artificial Sequence

<220>
<223> Pro CPB - Carboxypeptidase fusion protein

<220>
<221> PROPEP
<222> (1)..(402)
<223> X stands for

<400> 3

His Ser Gly Glu His Phe Glu Gly Asp Lys Val Phe Arg Val His Val
1 5 10 15

Glu Asp Glu Asn His Ile Ser Leu Leu His Glu Leu Ala Ser Thr Arg
20 25 30

Gln Met Asp Phe Trp Lys Pro Asp Ser Val Thr Gln Val Lys Pro His
35 40 45

Ser Thr Val Asp Phe Arg Val Lys Ala Glu Asp Thr Val Ala Val Glu
50 55 60

Asp Phe Leu Gly Gln Asn Gly Leu Arg Tyr Glu Val Leu Ile Ser Asn
65 70 75 80

Leu Arg Ser Met Leu Glu Ala Gln Phe Asp Ser Arg Val Arg Thr Thr
85 90 95

Gly His Ser Tyr Glu Lys Tyr Asn Asn Trp Glu Thr Ile Glu Ala Trp
100 105 110

Thr Glu Gln Val Ala Ser Glu Asn Pro Asp Leu Ile Ser Arg Ser Ala
115 120 125

Ile Gly Thr Thr Phe Leu Gly Asn Thr Ile Tyr Leu Leu Lys Val Gly
130 135 140

Lys Pro Gly Ser Asn Lys Pro Ala Val Phe Met Asp Cys Gly Phe His
145 150 155 160

Ala Arg Glu Trp Ile Ser Pro Ala Phe Cys Gln Trp Phe Val Arg Glu
165 170 175

Ala Val Arg Thr Tyr Gly Arg Glu Ile His Met Thr Glu Phe Leu Asp
180 185 190

Lys Leu Asp Phe Tyr Val Leu Pro Val Val Asn Ile Asp Gly Tyr Ile
195 200 205

Tyr Thr Trp Thr Thr Asn Arg Met Trp Arg Lys Thr Arg Ser Thr Arg
210 215 220

Ala Gly Ser Ser Cys Thr Gly Thr Asp Leu Asn Arg Asn Phe Asp Ala
225 230 235 240

Gly Trp Cys Ser Ile Gly Ala Ser Asn Asn Pro Cys Ser Glu Thr Tyr
245 250 255

Cys Gly Ser Ala Ala Glu Ser Glu Lys Glu Ser Lys Ala Val Ala Asp
260 265 270

Phe Ile Arg Asn His Leu Ser Ser Ile Lys Ala Tyr Leu Thr Ile His
275 280 285

Ser Tyr Ser Gln Met Met Leu Tyr Pro Tyr Ser Tyr Asp Tyr Lys Leu
290 295 300

Pro Lys Asn Asn Val Glu Leu Asn Thr Leu Ala Lys Gly Ala Val Lys
305 310 315 320

Lys Leu Ala Ser Leu His Gly Thr Thr Tyr Thr Tyr Gly Pro Gly Ala
325 330 335

Ser Thr Ile Tyr Pro Ala Ser Gly Gly Ser Asp Asp Trp Ala Tyr Asp
340 345 350

Gln Gly Ile Lys Tyr Ser Phe Thr Phe Glu Leu Arg Asp Lys Gly Arg
355 360 365

Tyr Gly Phe Val Leu Pro Glu Ser Gln Ile Gln Pro Thr Cys Glu Glu
370 375 380

Thr Met Leu Ala Ile Lys Tyr Val Thr Ser Tyr Val Leu Glu His Leu
385 390 395 400

Tyr Xaa

<210> 4
<211> 690
<212> DNA
<213> Artificial Sequence

<220>
<223> Nucleotide sequence of Trypsin gene

<220>
<221> gene
<222> (1)..(690)

<400> 4
gttgatgatg acgacaagat cgttggtggt tacacttgtg gtgctaacac cgttccatac 60

caggtttctt tgaactccgg ttaccacttc tgtggtggtt ccttgattaa ctcccagtgg 120

gttgtttctg ctgcccactg ttacaagtcc ggtatccaag ttagactggg tgaggacaac 180

atcaacgttg ttgaaggtaa cgagcagttc atctccgctt ccaagtctat cgttcaccca 240

tcctacaact ccaacaccct gaacaacgac atcatgctga tcaagttgaa gtccgctgct 300

tccttgaact ccagagttgc ttctatctcc ttgccaactt cttgtgcttc cgctggtact 360

cagtgtttga tttctggttg gggtaacacc aagtcctccg gtacttctta cccagacgtt 420

ttgaagtgtc tgaaggcccc aattttgtcc gactcttctt gtaagtctgc ctacccaggt 480

cagatcacct ccaacatgtt ttgtgctggt tacttggagg gtggtaagga ctcttgtcaa 540

ggtgattctg gtggtccagt tgtttgctcc ggtaagttgc aaggtatcgt ttcttggggt 600

tccggttgtg cccaaaagaa caagccaggt gtttacacca aggtctgcaa ctacgtttcc 660

tggatcaagc agactatcgc ctccaactaa 690

<210> 5
<211> 324
<212> PRT
<213> Artificial Sequence

<220>
<223> ProCPB Trypsin fusion protein

<220>
<221> PROPEP
<222> (1)..(324)
<223> X stands for

<400> 5

His Ser Gly Glu His Phe Glu Gly Asp Lys Val Phe Arg Val His Val
1 5 10 15

Glu Asp Glu Asn His Ile Ser Leu Leu His Glu Leu Ala Ser Thr Arg
20 25 30

Gln Met Asp Phe Trp Lys Pro Asp Ser Val Thr Gln Val Lys Pro His
35 40 45

Ser Thr Val Asp Phe Arg Val Lys Ala Glu Asp Thr Val Ala Val Glu
50 55 60

Asp Phe Leu Gly Gln Asn Gly Leu Arg Tyr Glu Val Leu Ile Ser Asn
65 70 75 80

Leu Arg Ser Met Leu Glu Ala Gln Phe Asp Ser Arg Val Arg Val Asp
85 90 95

Asp Asp Asp Lys Ile Val Gly Gly Tyr Thr Cys Gly Ala Asn Thr Val
100 105 110

Pro Tyr Gln Val Ser Leu Asn Ser Gly Tyr His Phe Cys Gly Gly Ser
115 120 125

Leu Ile Asn Ser Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser
130 135 140

Gly Ile Gln Val Arg Leu Gly Glu Asp Asn Ile Asn Val Val Glu Gly
145 150 155 160

Asn Glu Gln Phe Ile Ser Ala Ser Lys Ser Ile Val His Pro Ser Tyr
165 170 175

Asn Ser Asn Thr Leu Asn Asn Asp Ile Met Leu Ile Lys Leu Lys Ser
180 185 190

Ala Ala Ser Leu Asn Ser Arg Val Ala Ser Ile Ser Leu Pro Thr Ser
195 200 205

Cys Ala Ser Ala Gly Thr Gln Cys Leu Ile Ser Gly Trp Gly Asn Thr
210 215 220

Lys Ser Ser Gly Thr Ser Tyr Pro Asp Val Leu Lys Cys Leu Lys Ala
225 230 235 240

Pro Ile Leu Ser Asp Ser Ser Cys Lys Ser Ala Tyr Pro Gly Gln Ile
245 250 255

Thr Ser Asn Met Phe Cys Ala Gly Tyr Leu Glu Gly Gly Lys Asp Ser
260 265 270

Cys Gln Gly Asp Ser Gly Gly Pro Val Val Cys Ser Gly Lys Leu Gln
275 280 285

Gly Ile Val Ser Trp Gly Ser Gly Cys Ala Gln Lys Asn Lys Pro Gly
290 295 300

Val Tyr Thr Lys Val Cys Asn Tyr Val Ser Trp Ile Lys Gln Thr Ile
305 310 315 320

Ala Ser Asn Xaa

<210> 6
<211> 1677
<212> DNA
<213> Artificial Sequence

<220>
<223> Nucleotide sequence of Kexin gene

<220>
<221> gene
<222> (1)..(1677)

<400> 6
gaagctgaag ctgctccacc aatggattca tctttgttgc cagtaaaaga agccgaagat 60

aagttgtcca ttaacgaccc tttgttcgaa agacaatggc atttggttaa tccatctttc 120

ccaggttccg atatcaacgt tttggatttg tggtacaaca acattactgg tgctggtgtt 180

gttgctgcta tagttgatga tggtttggac tacgaaaacg aagatttgaa ggataacttc 240

tgcgctgaag gttcttggga ttttaacgat aacactaact tgccaaagcc aagattgtcc 300

gatgattatc atggtactag atgcgctggt gaaattgctg ctaaaaaggg taacaatttc 360

tgcggtgttg gtgttggtta caacgctaaa atttccggta tcagaatctt gtccggtgat 420

attactactg aagatgaagc tgcttctttg atctacggtt tggatgttaa cgatatctac 480

tcttgttctt ggggtccagc tgatgatggt agacacttgc aaggtccatc tgatttggtt 540

aagaaagctt tggttaaggg tgttaccgaa ggtagagatt ctaaaggtgc tatctacgtt 600

tttgcttctg gtaatggtgg tactagaggt gataactgta attacgatgg ttacaccaac 660

tccatctact ccattactat tggtgccatt gatcataagg acttgcatcc accatattct 720

gaaggttgtt ctgctgttat ggctgttact tattcttctg gttccggtga gtatatccac 780

tcctctgata ttaacggtag atgctctaat tctcacggtg gtacatctgc tgctgctcca 840

ttggctgctg gtgtttacac tttgttgttg gaagctaatc caaacttgac ttggagagat 900

gtccaatact tgtccatttt gtctgctgtt ggtttggaaa agaatgctga tggtgattgg 960

agagattctg ctatgggtaa aaagtactct cacagatacg gtttcggtaa gattgatgcc 1020

cataagttga tcgaaatgtc taagacttgg gaaaacgtta acgctcaaac ctggttttac 1080

ttgccaacct tgtatgtttc tcaatccact aactctaccg aagaaacctt ggaatccgtt 1140

attaccatct ccgaaaagtc attgcaagat gccaacttca agagaatcga acatgttact 1200

gttaccgttg atatcgacac cgaaattaga ggtactacta ccgttgattt gatttcccca 1260

gctggtatta tttccaactt gggtgttgtt agaccaagag atgtttcttc tgaaggtttc 1320

aaggattgga cctttatgtc tgttgctcat tggggtgaaa atggtgttgg tgattggaaa 1380

atcaaggtta agactaccga aaacggtcac agaatcgatt ttcattcttg gagattgaag 1440

ttgttcggtg aatccatcga ttcttctaag actgaaactt tcgttttcgg taacgacaaa 1500

gaagaagttg aaccagctgc tactgaatct actgtttctc aatattctgc ctcctccacc 1560

tccatttcta tttctgctac ttctacctcc tccatctcta ttggtgttga aacttctgct 1620

attccacaaa ctactactgc ttctactgat ccagattctg atccaaacac tccatga 1677

<210> 7
<211> 655
<212> PRT
<213> Artificial Sequence

<220>
<223> ProCPB Kexin fusion protein

<220>
<221> PROPEP
<222> (1)..(655)
<223> X stands for

<400> 7

His Ser Gly Glu His Phe Glu Gly Asp Lys Val Phe Arg Val His Val
1 5 10 15

Glu Asp Glu Asn His Ile Ser Leu Leu His Glu Leu Ala Ser Thr Arg
20 25 30

Gln Met Asp Phe Trp Lys Pro Asp Ser Val Thr Gln Val Lys Pro His
35 40 45

Ser Thr Val Asp Phe Arg Val Lys Ala Glu Asp Thr Val Ala Val Glu
50 55 60

Asp Phe Leu Gly Gln Asn Gly Leu Arg Tyr Glu Val Leu Ile Ser Asn
65 70 75 80

Leu Arg Ser Met Leu Glu Ala Gln Phe Asp Ser Arg Val Arg Asp Asp
85 90 95

Asp Asp Lys Arg Ala Pro Pro Met Asp Ser Ser Leu Leu Pro Val Lys
100 105 110

Glu Ala Glu Asp Lys Leu Ser Ile Asn Asp Pro Leu Phe Glu Arg Gln
115 120 125

Trp His Leu Val Asn Pro Ser Phe Pro Gly Ser Asp Ile Asn Val Leu
130 135 140

Asp Leu Trp Tyr Asn Asn Ile Thr Gly Ala Gly Val Val Ala Ala Ile
145 150 155 160

Val Asp Asp Gly Leu Asp Tyr Glu Asn Glu Asp Leu Lys Asp Asn Phe
165 170 175

Cys Ala Glu Gly Ser Trp Asp Phe Asn Asp Asn Thr Asn Leu Pro Lys
180 185 190

Pro Arg Leu Ser Asp Asp Tyr His Gly Thr Arg Cys Ala Gly Glu Ile
195 200 205

Ala Ala Lys Lys Gly Asn Asn Phe Cys Gly Val Gly Val Gly Tyr Asn
210 215 220

Ala Lys Ile Ser Gly Ile Arg Ile Leu Ser Gly Asp Ile Thr Thr Glu
225 230 235 240

Asp Glu Ala Ala Ser Leu Ile Tyr Gly Leu Asp Val Asn Asp Ile Tyr
245 250 255

Ser Cys Ser Trp Gly Pro Ala Asp Asp Gly Arg His Leu Gln Gly Pro
260 265 270

Ser Asp Leu Val Lys Lys Ala Leu Val Lys Gly Val Thr Glu Gly Arg
275 280 285

Asp Ser Lys Gly Ala Ile Tyr Val Phe Ala Ser Gly Asn Gly Gly Thr
290 295 300

Arg Gly Asp Asn Cys Asn Tyr Asp Gly Tyr Thr Asn Ser Ile Tyr Ser
305 310 315 320

Ile Thr Ile Gly Ala Ile Asp His Lys Asp Leu His Pro Pro Tyr Ser
325 330 335

Glu Gly Cys Ser Ala Val Met Ala Val Thr Tyr Ser Ser Gly Ser Gly
340 345 350

Glu Tyr Ile His Ser Ser Asp Ile Asn Gly Arg Cys Ser Asn Ser His
355 360 365

Gly Gly Thr Ser Ala Ala Ala Pro Leu Ala Ala Gly Val Tyr Thr Leu
370 375 380

Leu Leu Glu Ala Asn Pro Asn Leu Thr Trp Arg Asp Val Gln Tyr Leu
385 390 395 400

Ser Ile Leu Ser Ala Val Gly Leu Glu Lys Asn Ala Asp Gly Asp Trp
405 410 415

Arg Asp Ser Ala Met Gly Lys Lys Tyr Ser His Arg Tyr Gly Phe Gly
420 425 430

Lys Ile Asp Ala His Lys Leu Ile Glu Met Ser Lys Thr Trp Glu Asn
435 440 445

Val Asn Ala Gln Thr Trp Phe Tyr Leu Pro Thr Leu Tyr Val Ser Gln
450 455 460

Ser Thr Asn Ser Thr Glu Glu Thr Leu Glu Ser Val Ile Thr Ile Ser
465 470 475 480

Glu Lys Ser Leu Gln Asp Ala Asn Phe Lys Arg Ile Glu His Val Thr
485 490 495

Val Thr Val Asp Ile Asp Thr Glu Ile Arg Gly Thr Thr Thr Val Asp
500 505 510

Leu Ile Ser Pro Ala Gly Ile Ile Ser Asn Leu Gly Val Val Arg Pro
515 520 525

Arg Asp Val Ser Ser Glu Gly Phe Lys Asp Trp Thr Phe Met Ser Val
530 535 540

Ala His Trp Gly Glu Asn Gly Val Gly Asp Trp Lys Ile Lys Val Lys
545 550 555 560

Thr Thr Glu Asn Gly His Arg Ile Asp Phe His Ser Trp Arg Leu Lys
565 570 575

Leu Phe Gly Glu Ser Ile Asp Ser Ser Lys Thr Glu Thr Phe Val Phe
580 585 590

Gly Asn Asp Lys Glu Glu Val Glu Pro Ala Ala Thr Glu Ser Thr Val
595 600 605

Ser Gln Tyr Ser Ala Ser Ser Thr Ser Ile Ser Ile Ser Ala Thr Ser
610 615 620

Thr Ser Ser Ile Ser Ile Gly Val Glu Thr Ser Ala Ile Pro Gln Thr
625 630 635 640

Thr Thr Ala Ser Thr Asp Pro Asp Ser Asp Pro Asn Thr Pro Xaa
645 650 655

<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Forward primer seqeunce

<220>
<221> misc_feature
<222> (1)..(21)

<400> 8
tactattgcc agcattgctg c 21

<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence

<220>
<223> Reverse Primer Sequence

<220>
<221> misc_feature
<222> (1)..(21)

<400> 9
gcaaatggca ttctgacatc c 21
,CLAIMS:1. A fusion protein comprising propeptide sequence of Carboxypeptidase B; and a Protease.

2. The fusion protein as claimed in claim 1, wherein the protease is selected from the group comprising Enterokinase, Lysine endopeptidase, Asp N endoproteinase Carboxypeptidase B, Trypsin and Kexin.

3. The fusion protein as claimed in claim 1, wherein the fusion protein comprises amino acid sequence selected from the group comprising SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7.

4. A recombinant nucleic acid molecule encoding the fusion protein of claim 1.

5. A vector comprising the recombinant nucleic acid molecule of claim 4.

6. A host cell comprising the vector of claim 5.

7. The host cell as claimed in claim 6, wherein the host cell is selected from the group comprising – E.coli, Saccharomyces cerevisiae, Hansenula, Bacillus, Yarrowia Kluveromyces, and Pichia pastoris.

8. A process for producing a recombinant protease, said process comprising steps of:
a) Introducing a vector which comprises recombinant nucleic acid molecule of claim 4 and encoding the fusion protein of claim 1, into a host cell;
b) Culturing the host cell to produce the fusion protein; and
c) Cleaving the fusion protein to produce the recombinant protease.

9. The method as claimed in claim 8, wherein the fusion protein is cleaved by method selected from the group comprising autocatalytic cleavage, change in pH, increased temperature, active enzymes and combinations thereof.

10. A process of converting protein precursor selected from the group comprising Insulin, Glargine, Aspart, Lispro, Glulisine, Insulin, Detemir and combinations thereof, into active protein, said process comprising act of contacting the protein precursor with protease selected from the group comprising Carboxypeptidase B, Trypsin, Kexin, Enterokinase, Asp N endoproteinase and Lysine endopeptidase.

11. The process as claimed in claim 10, wherein the protease is the recombinant protease produced by the process of claim 8.