FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL SPECIFICATION
(See Section 10 and Rule 13)
CLONING, EXPRESSION, PURIFICATION AND l/SE OF RECOMBINANT
ENTEROKINASE
Intas Biopharmaceuticals Limited
An Indian company having its registered office at:
Plot No: 423/P/A/GIDC
Sarkhej-Bavla Highway
Moraiya, Tal.: Sanand
Ahmedabad - 382 213
Gujarat, India
The following specification describes the invention.

FIELD OF THE INVENTION
The present invention relates to, cloning, expression and production of recombinant human Enterokinase with a novel refolding process for better enzymatic activity,
BACKGROUND OF THE INVENTION
Enterokinase (EK) also known as Enteropeptidase is a heterodimeric serine protease enzyme which converts inactive trypsinogen into active trypsin. The native enzyme has a heavy chain (~115kDa) linked by a disulfide bond to a light chain or catalytic subunit (35kDa). The enzyme possesses high specificity and cleaves after amino acid Lysine if the Lysine is preceded by 4 Asparatic acid residues and not followed by Proline. This enzyme shows good activity in the presence of various detergents and denaturants over a wide pH range (4.5-9.5) and temperature range (4-45°C). Therefore, EK is considered as a powerful tool widely applicable in biotechnology.
Literature review shows that recombinant expression of EK light chain in Escherichia coli (£. coli) and yeast is tried by various groups but biologically active EK is not easy to produce. Various groups including, Taylor A et al, (Microbiology and Biotechnology, 1992: 37(2) 205-210), La Vallie et al, (Biotechnology 1995; 13, 982-987) has tried expressing EK in E. coli and could not get active protein.
Other groups including Gasparian et al, (Protein Expression and Purification 2003: 31, 133-139), Huang et al, (Chinese Medical Journal 2003: 117 (2), 286-290), La Vallie et al, (Journal of Biological Chemistry, 1993: 31, 23311-23317) have used fusion partners to express EK in the soluble active form.
Collins Racie et al, (Biotechnol. 1995, vol. 13, pp. 982-987) reported the production of recombinant bovine enterokinase in Escherichia coli at a very low level using a novel secretory fusion partner DsbA Oxidoreductase.
Sadler et al, (Biochem. 1995, vol. 34, pp. 4562-4568) isolated the cDNA from a human cDNA library that encoded the complete human enterokinase amino acid sequence from a human intestine cDNA library. Starting from the ATG codon, the composite 3696 nt cDNA sequence contains an open reading frame of 3057 nt that encodes 784 amino acid heavy chain followed by

a 235 amino acid light chain; the two chains are linked by at least one disulfide bond. The heavy chain contains a potential N-terminal myristoylation site, a potential signal anchor sequence near amino terminus, and six structural motifs that are found in otherwise unrelated proteins. These Structural features are conserved among human, bovine and porcine enterokinase. By Northern blotting, a 4.4kb enterokinase mRNA was detected only in small intestine. The EK gene was localized to human chromosome 21 q21 by fluorescence in situ hybridization.
US5665566 described about nucleic acid sequences encoding bovine EK, the expression product thereof, and methods for using the same.
US6746859 described about a recombinant, biologically active, EK light chain, free of EK heavy chain, comprising amino acids 564 to 798 of SEQ ID NO: 2
The light chain of human EK has 9 Cysteine residues which make 4 intra chain disulphide linkages and atieast one disulphide bond with the heavy chain. For biological activity of the protein, proper folding of the protein is essential and disulphide bonds play a critical role in proper folding of EK. To overcome the existing problems and to get properly folded recombinant EK with better enzymatic activity, we have developed a new process for cloning, refolding and purification of the EK which helped us to recover more of the properly folded and biologically active form of EK.
SUMMARY OF THE INVENTION
A plasmid comprising a nucleic acid sequence of SEQ. ED. No. 1 encodes for an enzyme, EK having an amino acid sequence as shown in SEQ. ID. No. 2 wherein N-terminal of the said SEQ.ID. No. 1 is operably linked to EK cleavage recognition site, poly histidine tag, signal sequence, start codon and an inducible promoter.
A host cell carrying the recombinant vector comprising the nucleic acid sequence as mentioned above.
A process for the production of Enterokinase having an amino acid sequence of SEQ, ID. No. 2, wherein said method comprising:

• Culturing the host cells in a growth medium by maintaining specific culture conditions
• Purifying Enterokinase
• Refolding and recovering Enterokinase
A process for the purification of enterokinase produced as per the present invention, wherein the purification process comprisies the following steps:
• Affinity chromatography
• On column refolding and
• Recovery by autocatalytic activity
Enterokinase obtained according to the above mentioned process showed better enzymatic activity compared to the commercially available Enterokinase.
Use of EK produced from the above process to cleave proteins at a specific cleavage site essential for removing tags from recombinant fusion proteins.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Construct design: showing uniqueness of the construct with an N-terminal HIS tag and bacterial specific signal sequence along with Enterokinase cleavage recognition site.
Figure 2: Recombinant Vector showing the functional components of the expression machinery for Enterokinase protein expression.
Figure 3: Single step protein purification using N-terminal HIS tag.
DESCRIPTION OF THE INVENTION
The present invention relates to the cloning and expression of EK gene and an improved process for production, purification and refolding of EK enzyme using recombinant DNA technology. The method disclosed in the invention produces an active form of EK which is comparable to commercially available EK and widely used in industry to clean proteins at specific amino acid sequence DDDDKX wherein X is not a protein.

The present invention relates to a process for cloning, expression and purification of recombinant human enterokinase light chain.
Recombinant human enterokinase light chain produced according to the present invention was then purified from the inclusion bodies (IBs) by using a novel purification process, which gave better enzymatic activity compared to the commercially available Enterokinase.
One embodiment of the present invention relates to a plasmid comprising a nucleic acid sequence of SEQ, ID. No. 1 encodes for an enzyme, Enterokinase having an amino acid sequence as shown in SEQ. ID. No. 2 wherein N-terminal of the said SEQ.ID. No. 1 is operably linked to Enterokinase recognition site, polyhistidine tag, signal sequence, start codon and an inducible promoter.
Another embodiment of the present invention provides a recombinant vector carrying the nucleic acid sequence of SEQ. ID. No. 1.
Still another embodiment of the present invention, wherein the said inducible promoter is selected from a group consisting of T7, trp, lac and tac promoters.
Yet another embodiment of the present invention provides the host cell comprising the recombinant vector comprising the nucleic acid sequence of SEQ. ID. NO. 1, wherein the host cell is selected from the group consisting of prokaryotic and eukaryotic cell lines.
In yet another embodiment of the present invention provides the process for the production of enterokinase, wherein said method comprising:
• Culturing the host cells in a growth medium by maintaining specific culture conditions
• Purifying Enterokinase
• Refolding Enterokinase and
• Recovering Enterokinase •
In another embodiment of the present invention provides a process for the purification of enterokinase produced as described herein, wherein the purification process comprises the following steps:
• Affinity chromatography

• On column refolding
• Analyzing activity of the Enterokinase enzyme
The last embodiment of the present invention provides use of an enzyme obtained by the said purification process for cleaving fusion proteins produced by recombinant DNA technology to obtain purified proteins.
Examples/ Experiments
The following examples/ experiments will further illustrate certain aspects and embodiments of the invention in detail and not intended to limit the scope of the invention.
Example 1
Enterokinase gene was chemically synthesized with desired sequences as detailed in SEQ. ID No.l. Human EK gene coding for the light chain is 705 base pairs. The gene was synthesized with restriction enzyme sites which were used during the cloning of gene in expression vector to generate the final construct as shown in Figure 1. The final construct (Figure 2) was used to transform E. coli cells. The transformants were screened for the presence of gene insert and later on for the protein expression.
Example 2
Expression of Enterokinase in E.coli:
Positive clones carrying the gene of interest were inoculated in SY broth, containing ampicillin. The cultures were induced with EPTG and protein expression was checked using SDS-PAGE gels. The protein expression was optimized using parameters including IPTG concentration, temperature of growth, time of induction and cell culture density. A cell bank of the expressing clones was prepared.
Example 3 Fermentation Process
The upstream process for the recombinant E.coli clone harboring the enterokinase gene comprised of a new feeding strategy, constant feeding combined with pH-stat to avoid the accumulation of substrate in culture broth. Limiting amount of substrate was fed. When pH rose above an upper limit due to the depletion of principal carbon source, feeding was restarted.

Induction was done with IPTG at the late log phase once the desired biomass was achieved. With this feeding strategy, recombinant E. colt could be grown upto 110 g/1 with an IB yield of 12 g/1 by controlling the specific growth rate at 0.15.
Example 4
Purification and refolding process:
The protein was expressed in the form of IBs, which were solubilized in a buffer containing 6M Urea/Guanidine hydrochloride. The protein was dialyzed overnight to partially remove the denaturant before purification using Ni-NTA affinity chromatography (Figure 3). The tagged protein was captured on the column and the final round of refolding was done on the column itself. This folds the protein in its native form and induces autocatalytic cleavage of the fusion tag from N-terminal of the protein. We have collected the fully refolded protein in the flow through of the column.
Example 5
Enzymatic activity analysis:
In vitro enzymatic assay of recombinant human EK was measured by its ability to cleave a colorimetric peptide substrate, N-carbobenzyloxy-Lys-ThioBenzyl easter (Z-Lys-SBzl), in the presence of 5, 5' Dithio-bis 2 nitrobenzoic acid (DTNB).
The assay was set up by adding 50 mcl of assay buffer to 50 mcl of refolded EK and commercially available EK. The plate was read in kinetic mode at 405 nM for 10 minutes at one minute intervals. The rate of change in absorbance is directly proportional to the enzymatic activity.