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Details for Patent: 5,618,697

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Details for Patent: 5,618,697

Title: Process for preparing a desired protein
Abstract:A process for producing ripe human growth hormone is described which comprises introducing into bacteria a recombinant DNA vector comprising a DNA sequence encoding an amino-terminal extended human growth hormone which amino-terminal extension has a negatively charged amino acid sequence and has an even number of amino acids, growing the bacteria such that the amino-terminal extended human growth is expressed, separating the amino-terminal extended human grown hormone from contaminants, cleaving the amino-terminal extension with dipeptidyl aminopeptidase I to obtain ripe human growth hormone and isolating the ripe human growth hormone produced.
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 W. (Br.o slashed.ndby, DK), Jessen; Torben E. (Holbaek, DK)
Assignee: Novo Nordisk A/S (DK)
Filing Date:Jan 13, 1995
Application Number:08/372,692
Claims:1. 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 a negatively charged amino acid sequence having an even number of amino acids, in which the amino acid sequence X contains no more than 4 amino acids and 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 the C-terminal amino acid of X is a negatively 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) from uncleaved amino-terminal extended human growth hormone and partially cleaved amino-terminal extended human growth hormone on the basis of the difference in the net electrical charge between said ripe human growth hormone and said uncleaved and partially cleaved amino-terminal extended human growth hormone.

2. The process according to claim 1 in which the amino acid sequence X contains no more than two amino acids.

3. The process according to claim 2 in which the C-terminal amino acid of X is Glu or Asp.

4. The process according to claim 1 in which the N-terminal amino acid of X is Met.

5. The process according to claim 4 in which the C-terminal amino acid of X is Glu or Asp.

6. The process according to claim 1 in which the C-terminal amino acid of X is Glu or Asp.

7. The process according to claim 1 in which X is Ala-Glu.

8. The process according to claim 1 in which the amino acid sequence X is Met-Glu-Ala-Glu.

9. The process according to claim 1 in which the amino acid sequence X is Met-Phe-Glu-Glu.

10. The process according to claim 1 in which the amino acid sequence X is Met-Thr-Glu-Glu.

11. The process according to claim 1 in which the amino acid sequence X is Met-Glu-Glu-Glu.

12. The process according to claim 1 in which the amino acid sequence X is Ala-Glu-Ala-Glu.
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