Patent
Anti diabetic protein
| العنوان: | Anti diabetic protein |
|---|---|
| Patent Number: | 8,669,348 |
| تاريخ النشر: | March 11, 2014 |
| Appl. No: | 12/594007 |
| Application Filed: | March 28, 2008 |
| مستخلص: | The present invention relates to a novel hypoglycemic/anti-hyperglycemic protein named ADMc1 purified from the seeds of Momordica charantia for control of hyperglycemia. The process for the purification of novel hypoglycemic/anti-hyperglycemic protein named ADMc1 is also disclosed. The invention also relates to process for preparation and purification of the recombinant novel hypoglycemic/anti-hyperglycemic protein of Momordica charantia, named rADMc1. Both ADMc1 and rADMc1 are highly effective and need to be administered only once a day to maintain normal blood glucose levels. The procedure involves purification of a novel hypoglycemic/anti-hyperglycemic protein of M. charantia, construction of cDNA library from M. charantia seeds, screening of cDNA library using oligonucleotide probe designed on the basis of amino acid sequence of the tryptic fragment of the protein, cloning of the cDNA in a eukaryotic expression system, expression and purification of the recombinant protein. |
| Inventors: | Dixit, Aparna (New Delhi, IN); Vashishta, Aruna (New Delhi, IN); Sahu, Tejram (New Delhi, IN); Choudhary, Shailesh Kumar (New Delhi, IN); Murugesan, Alli (New Delhi, IN) |
| Assignees: | Dixit, Aparna (New Delhi, IN) |
| Claim: | 1. A hypoglycemic/anti-hyperglycemic protein comprising ADMc1 purified from seeds of Momordica charantia consisting of SEQ ID NO:2. |
| Claim: | 2. A composition as claimed in claim 1 , consisting of hypoglycemic/anti-hyperglycemic protein consisting of SEQ ID NO:2 and one or more excipients, vehicles, additives, adjuvants or modulators. |
| Claim: | 3. A hypoglycemic/anti-hyperglycemic protein consisting of two chains of ˜4 kDa consisting of SEQ ID NO:3 and ˜8 kDa consisting of SEQ ID NO:4. |
| Claim: | 4. A composition comprising a hypoglycemic/anti-hyperglycemic protein, wherein said hypoglycemic/anti-hyperglycemic protein comprises ADMc 1 or recombinant ADMc 1 wherein said ADMc 1 or recombinant ADMc 1 comprises residues 37 to 66 and 73 to 138 of SEQ ID NO: 6. |
| Claim: | 5. A cDNA consisting of SEQ ID NO:5 encoding hypoglycemic/antihyperglycemic protein ADMc 1 of Momordica charantia. |
| Claim: | 6. A process for the purification of hypoglycemic/anti-hyperglycemic protein ADMc 1 consisting of SEQ ID NO: 2 purified from seeds of Momordica charantia comprising the steps of: a) decorticating and powdering said seeds; b) extracting seed proteins from the decorticated and powdered seeds obtained from step (a); c) fractionating said seed proteins; d) separating a soluble protein fraction and pelleted proteins wherein the soluble protein fraction is enriched in hypoglycemic/anti-hyperglycemic protein ADMc 1 consisting of SEQ ID NO: 2; and e) purifying the protein fraction enriched in hypoglycemic/anti-hyperglycemic activity to homogeneity. |
| Claim: | 7. The process as claimed in claim 6 , wherein performing said decorticating and powdering of step a) occurs in liquid nitrogen. |
| Claim: | 8. The process as claimed in claim 6 , wherein performing said extracting seed proteins of step b) via a mixture of buffered sulfuric acid ethanol or other acceptable extraction medium, containing protease inhibitor phenylmethylsulfonyl fluoride. |
| Claim: | 9. The process as claimed in claim 8 , wherein said phenylmethylsulfonyl fluoride is 0.5-3 mM. |
| Claim: | 10. The process for the purification of hypoglycemic/anti-hyperglycemic protein as claimed in claim 6 , wherein performing said fractioning of step c) via centrifuging at 16,000-20,000×g for 1-2 h at 4°, by ultrafiltration, by salt or pH gradient or a combination thereof. |
| Claim: | 11. The process for the purification of hypoglycemic/anti-hyperglycemic protein as claimed in claim 6 , wherein performing said purifying said protein fraction enriched in hypoglycemic/anti-hyperglycemic protein ADMc1 consisting of SEQ ID NO:2 of step e) via subjecting the soluble protein fraction to reverse-phase HPLC. |
| Claim: | 12. A process for preparation and purification of recombinant hypoglycemic/anti-hyperglycemic protein rADMc1 purified from seeds of Momordica charantia comprising the steps of: (i) isolating and purifying mRNA from unripe fruits bearing developing seeds of M. charantia; (ii) preparing cDNA from said purified mRNA; (iii) isolating the cDNA of SEQ ID NO:5 from cDNA prepared in step (ii) encoding said hypoglycemic/antihyperglycemic protein; (iv) cloning the isolated cDNA consisting of SEQ ID NO:5 encoding said hypoglycemic/anti-hyperglycemic protein into an expression vector; (v) isolating cDNA consisting of SEQ ID NO:5 encoding said hypoglycemic/anti-hyperglycemic protein; (vi) transforming Pichia pastoris with said cDNA of step (v); (vii) cultivating Pichia pastoris containing said hypoglycemic/anti-hyperglycemic protein clone wherein said hypoglycemic/anti-hyperglycemic protein clone is cultivated by culturing said Pichia pastoris in culture medium to produce recombinant hypoglycemic/anti-hyperglycemic protein rADMc1; and (viii) purifying said recombinant hypoglycemic/anti-hyperglycemic protein from said culture medium. |
| Claim: | 13. The process as claimed in claim 12 , wherein said Pichia pastoris is Pichia pastoris GS115. |
| Claim: | 14. The process as claimed in claim 12 , wherein said expression vector is pPIC9K. |
| Claim: | 15. The process as claimed in claim 12 , wherein said culture medium is a combination of growth culture medium and induction culture medium. |
| Claim: | 16. The process as claimed in claim 15 , wherein said growth culture medium is buffered minimal glycerol (BMG) medium. |
| Claim: | 17. The process as claimed in claim 15 , wherein said induction culture medium is buffered minimal methanol (BMM) medium. |
| Claim: | 18. The process as claimed in claim 12 , wherein said purifying of step (viii) occurs by one-step size exclusion chromatography. |
| Claim: | 19. The process as claimed in claim 12 , comprising obtaining from one liter of induction culture about 70 mg of purified protein. |
| Claim: | 20. A method of treating a mammal suffering from hyperglycemia which comprises administering to said mammal hypoglycemic/anti-hyperglycemic protein consisting of SEQ ID NO: 2 or recombinant ADMc 1 comprising residues 37 to 66 and 73 to 138 of SEQ ID NO: 6, or a pharmaceutical composition comprising said hypoglycemic/anti-hyperglycemic protein consisting of SEQ ID NO: 2 or recombinant ADMc 1 comprising residues 37 to 66 and 73 to 138 of SEQ ID NO: 6. |
| Claim: | 21. The method as claimed in claim 20 , wherein said mammal is a human. |
| Claim: | 22. The method as claimed in claim 20 , wherein administering is via a single dose of said hypoglycemic/anti-hyperglycemic protein so as to reduce the blood glucose levels of a diabetic subject by 50-80% and maintains the lower blood glucose level for up to 24 hrs. |
| Claim: | 23. The method as claimed in claim 20 , wherein the single dose of 5 to 15 mg/kg body weight per day does not show any toxic side effect in the mammal. |
| Current U.S. Class: | 530/370 |
| Patent References Cited: | 6831162 December 2004 Khanna 00/61619 October 2000 |
| Other References: | Vashishta et al., 2006, In vitro refolded napin-like protein of Momordica charantia expressed in Escherichia coli displays properties of native napin, Biochimica et Biophysica Acta, 1764: 847-855. cited by examiner Neumann et al., 1996, Purification and sequencing of napin-like protein small and large chains from Momordica charantia and Ricinus communis seeds and determination of sites phosphorylated by plant Ca2+-dependent protein kinase, Biochimica et Biophysica Acta, 1298: 223-240. cited by examiner Terras, Franky R. G., et al. “A new family of basic cysteine-rich plant antifungal proteins from Brassicaceae species” FEBS Letters, vol. 316, No. 3, 233-240 (Feb. 1993). cited by applicant |
| Primary Examiner: | Steele, Amber D |
| Attorney, Agent or Firm: | Ladas & Parry LLP |
| رقم الانضمام: | edspgr.08669348 |
| قاعدة البيانات: | USPTO Patent Grants |
| الوصف غير متاح. |