.

Pharmaceutical Business Intelligence

  • Anticipate P&T budget requirements
  • Evaluate market entry opportunities
  • Find generic sources and suppliers
  • Predict branded drug patent expiration

► Plans and Pricing

Upgrade to enjoy subscriber-only features like email alerts and data export. See the Plans and Pricing

DrugPatentWatch Database Preview

Details for Patent: 5,691,169

« Back to Dashboard

Details for Patent: 5,691,169

Title: Process for preparing a desired protein
Abstract:A desired protein having the formula: wherein a) A is Lys or Arg, and B and C are arbitrary amino acids, or b) A is an arbitrary amino acid different from Pro, Lys and Arg, and B and/or C is Pro, is produced from a biosynthetically formed amino acid extended protein having the formula: wherein A, B, C and P are as defined above, and X is an amino acid sequence with an even number of amino acids, of which the first one, seen from the N-terminal end, is different from Lys and Arg, all other uneven amino acids are different from Pro, Lys and Arg, and all even amino acids are different from Pro, by reaction with the enzyme dipeptidyl aminopeptidase (DAP I). The desired protein is obtained in a pure state. Thus, e.g. hGH without content of Met-hGH may be produced by the process.
Inventor(s): Dalb.o slashed.ge; Henrik (Virum, DK), Pedersen; John (Kokkedal, DK), Christensen; Thorkild (Aller.o slashed.d, DK), Ringsted; J.o slashed.rli Winnie (Br.o slashed.ndby, DK), Jessen; Torben Ehlern (Holbaek, DK)
Assignee: Novo Nordisk A/S (DK)
Filing Date:Mar 10, 1995
Application Number:08/402,455
Claims:1. A process for preparing ripe proteins, which comprises expressing in a procaryotic or eukaryotic cell a DNA sequence which codes for the synthesis of a fusion protein which comprises the amino acid sequence of the desired ripe protein and a N-terminal amino acid pro-sequence, cleaving the pro-sequence using DAP I and recovering the desired ripe protein.

2. The process according to claim 1 wherein the cell is a bacterial cell.

3. The process according to claim 2 wherein the bacterial cell is E. coli.

4. The process according to claim 1 wherein the ripe protein is a peptide hormone.

5. The process according to claim 1 wherein the desired ripe protein is human growth hormone and the pro-sequence is negatively charged.

6. The process according to claim 1 wherein the amino acid sequence of the pro-sequence is selected to facilitate purification of the fusion protein.

7. The process according to claim 1 wherein the amino acid sequence of the pro-sequence is selected so that the desired cleavage using DAP-I is facilitated.

8. The process according to claim 1 wherein the amino acid sequence of the pro-sequence is selected to facilitate transportation of the fusion protein out of the cell and extracellular cleavage by DAP-I.

9. The process according to claim 1 wherein the ripe protein has proline as the next-to-the-outermost N-terminal amino acid.

10. The process according to claim 1 in which the pro-sequence contains 2 amino acids.

11. The process according to claim 1 in which the pro-sequence is Met-Glu-Ala-Glu.

12. The process according to claim 1 in which the pro-sequence is Met-Phe-Glu-Glu.

13. The process according to claim 1 in which the pro-sequence is Met-Thr-Glu-Glu.

14. The process according to claim 1 in which the pro-sequence is Met-Glu-Glu-Glu.

15. The process according to claim 1 in which the pro-sequence is Ala-Glu-Ala-Glu.

16. The process according to claim 1 in which the initial amine acid of the pro-sequence is Met.

17. The process according to claim 1 in which the pro-sequence is Ala-Glu.

18. The process according to claim 1 in which the C-terminal amine acid of the pro-sequence is glu or Asp.

19. A process for separating a desired ripe protein from a fusion protein comprising the desired ripe protein having a negatively charged amino acid sequence of suitable length fused to the N-terminus of said desired ripe protein, said amino acid sequence comprising an even number of amino acids, which process comprises cleaving the amino acid sequence using dipeptidyl aminopeptidase I under suitable cleavage conditions, and recovering the desired ripe protein utilizing the existence of the negative charge in the amino acid sequence.

20. A process according to claim 19 wherein the amino acid sequence has an amino acid having a negative charge on the amino acid which is directly attached to the N-terminus of the ripe protein.

21. A process according to claim 19 wherein the amino acid sequence is four amino acids in length.

22. A process according to claim 19 wherein the amino acid sequence is two acid acids in length.

23. A process according to claim 19 wherein the C-terminal amino acid of the amino acid sequence is Glu or Asp.

24. A process according to claim 19 wherein the N-terminal amino acid of the amino acid sequence is Met.

25. A process according to claims 24 wherein the C-terminal amino acid of the amino acid sequence is Glu or Asp.

26. A process according to claim 19 wherein the amino acid sequence is Ala-Glu.

27. A process according to claim 19 wherein the amino acid sequence is Met-Glu-Ala-Glu.

28. A process according to claim 19 wherein the amino acid sequence is Met-Phe-Glu-Glu.

29. A process according to claim 19 wherein the amino acid sequence is Met-Thr-Glu-Glu.

30. A process according to claim 19 wherein the amino acid sequence is Met-Glu-Glu-Glu.

31. A process according to claim 19 wherein the amino acid sequence is Ala-Glu-Ala-Glu.

32. A process according to claim 19 wherein the ripe protein is human growth hormone.

33. A process according to claim 19 wherein the ripe protein is interlinear I.

34. A process according to claim 19 wherein the ripe protein is human lysozyme.

35. A process according to claim 19 wherein the ripe protein is insulin-like growth factor I.

36. A process according to claim 19 wherein the ripe protein is bovine growth hormone.

37. A process for separating a desired ripe protein from a fusion protein comprising the desired ripe protein having a charged amino acid sequence of suitable length fused to the N- terminus of said desired ripe protein wherein at least one of the charged amino acids is attached to the N-terminal of said desired ripe protein, said amino acid sequence comprising an even number of amino acids, which process comprises cleaving the amino acid sequence using dipeptidyl aminopeptidase I under suitable cleavage conditions, and recovering the desired ripe protein utilizing the existence of the charge in the charged amino acid sequence.

38. A process according to claim 37 wherein the charged amino acid sequence is either exclusively positively charged or exclusively negatively charged.

39. A process according to claim 37 wherein the charged amino acid sequence is exclusively positively charged.

40. A process according to claim 37 wherein the amino acid sequence is four amino acids in length.

41. A process according to claim 37 wherein the amino acid sequence is two amino acids in length.

42. A process for preparing a desired ripe protein which comprises expressing in a procaryotic or eukaryotic cell a DNA sequence which codes for the synthesis of a fusion protein which comprises the amino acid sequence of the desired ripe protein and an N-terminal amino acid pro-sequence, cleaving the pro-sequence using an dipeptidyl aminopeptidase and recovering the desired ripe protein.

43. A method of producing a protein consisting of amine acids 1-191 of human growth hormone comprising:

a) expressing in a bacterial cell, DNA coding for a human growth hormone conjugate protein, which conjugate protein consists of amino acids 1-191 of human growth hormone and the additional amino acid sequence Met-Glu-Ala-Glu, and

(b) cleaving extracellularly said conjugate protein using DAP I to produce said protein consisting of amino acid 1-191 of human growth hormone.

44. A process for producing ripe human growth hormone comprising

(a) introducing into bacteria a recombinant DNA vector comprising a DNA sequence encoding an amino-terminal extended human growth hormone having the formula

in which X is an amino acid sequence in which the N-terminal amino acid of X is different from Lys and Arg, all other uneven amino acids are different from Pro, Lys and Arg, and all even amino acids are different from Pro and in which at least one amino acid in the amino acid sequence X is a charged amino acid;

(b) growing the bacteria such that the amino-terminal extended human growth hormone is expressed;

(c) separating the amino-terminal extended human growth hormone in step (b) from contaminants;

(d) cleaving the amino-terminal extension of the amino-terminal extended human growth hormone of step (c) with dipeptidyl aminopeptidase I to obtain ripe human growth hormone; and

(e) isolating ripe human growth hormone from step (d).
« Back to Dashboard

For more information try a trial or see the database preview and plans and pricing

Drugs may be covered by multiple patents or regulatory protections. All trademarks and applicant names are the property of their respective owners or licensors. Although great care is taken in the proper and correct provision of this service, thinkBiotech LLC does not accept any responsibility for possible consequences of errors or omissions in the provided data. The data presented herein is for information purposes only. There is no warranty that the data contained herein is error free. thinkBiotech performs no independent verifification of facts as provided by public sources nor are attempts made to provide legal or investing advice. Any reliance on data provided herein is done solely at the discretion of the user. Users of this service are advised to seek professional advice and independent confirmation before considering acting on any of the provided information. thinkBiotech LLC reserves the right to amend, extend or withdraw any part or all of the offered service without notice.

`abc