MODIFIED SAK GENE FOR THE PRODUCTION OF RECOMBINANT PROTEINS

• US 20150010944A1
• Filed: 08/28/2014
• Published: 01/08/2015
• Est. Priority Date: 06/04/2010
• Status: Active Grant

• Current Assignee: Lupin Limited
• Original Assignee: Lupin Limited
• Inventors: SALUNKHE, Shardul, PRASAD, Bhaskarjyoti, MUNESHWAR, Praveen, DESHPANDE, Anjali Apte, PADMANABHAN, Sriram
• Granted Patent: US 9,670,262 B2
• Time in Patent Office: Days
• US Class Current: 435/68.1
• CPC Class Codes: C07K 14/31 from Staphylococcus (G)
• CPC Class Codes: C07K 14/485 Epidermal growth factor [EGF], i.e. urogastrone
• CPC Class Codes: C07K 14/49 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C07K 14/5403 IL-3
• CPC Class Codes: C07K 14/5406 IL-4
• CPC Class Codes: C07K 14/5409 IL-5
• CPC Class Codes: C07K 14/5412 IL-6
• CPC Class Codes: C07K 14/5431 IL-11
• CPC Class Codes: C07K 14/55 IL-2
• CPC Class Codes: C07K 14/555 Interferons [IFN]
• CPC Class Codes: C07K 14/635 Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
• CPC Class Codes: C07K 2319/35 containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C12N 9/6459 t-plasminogen activator (3.4.21.68), i.e. tPA
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00
• CPC Class Codes: C12Y 304/21069 Protein C activated (3.4.21.69)

Abstract

The present invention relates to modified SAK gene having amino acid SEQ ID 2. The present invention further relates to process for cloning and expressing modified SAK gene fusion protein which imparts improved stability to the heterologous protein of interest. Further the invention relates to process of purification of recombinant heterologous proteins from bacterial inclusion bodies using modified SAK.

Claims

1. A process for the preparation of heterologous protein of interest in E. coli comprising the steps of:

a) preparing a fusion DNA comprising a first DNA encoding modified SAK protein and a second DNA fused in the frame encoding the heterologous protein of interest,

b) cloning of the fusion DNA of step a into an expression vector,

c) expressing the fusion protein in E. coli cells,

d) optionally refolding and purifying the fusion protein;

e) enterokinase cleavage of fusion protein, and

f) isolating and purifying the protein of interest to obtain pure heterologous protein.

2. The process as claimed in claim 1, wherein the heterologous protein of interest is selected from the group comprising cytokines, growth stimulating factors, hormones, interferons, interleukins, enzymes.

3. The process as claimed in claim 1, wherein the heterologous protein of interest is selected from the group comprising parathyroid hormone (1-34), parathyroid hormone (1-84), reteplase, interferon, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, GCSF, epidermal growth factor and platelet derived growth factor.

4. The process as claimed in claim 1, wherein the heterologous protein of interest is parathyroid hormone (1-34).

5. The process as claimed in claim 1, wherein the heterologous protein of interest is IL-11.

6. A method for purification of modified SAK fusion protein wherein, the fusion protein is expressed as inclusion bodies in E coli comprising steps of:

a) solubilizing the inclusion bodies,

b) refolding the solubilizing inclusion bodies,

c) enterokinase digestion of modified SAK fusion protein, and

d) isolating and purifying the protein of interest by one or more of chromatographic techniques.

7. The method as claimed in claim 6, wherein the protein may be refolded before or after enterokinase digestion.

8. The method as claimed in claim 6, wherein the fusion protein can be purified by purification methods comprising Ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, reverse phase chromatography and gel filtration chromatography

9. The method as claimed in 6, wherein ion exchange chromatography is selected from cation exchange and anion exchange in any order.

10. The method as claimed in 8, wherein hydrophobic interaction chromatography is performed using resins selected from butyl sepharose, phenyl sepharose or octyl sepharose.