# US 5,352,769 A · Enzymatic removal of a protein amino-terminal sequence

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- Number: US 5,352,769 A
- Document ID: US5352769A
- Status: Granted
- Kind: A
- Application number: 07/864,098
- Filed: 1992-04-06
- Granted: 1994-10-04
- Earliest claimed: 1989-05-09
- Assignees: Eli Lilly and Company
- Inventors: Gerald W. Becker; Thomas C. Furman; Warren C. MacKellar; James P. McDonough
- Web page: https://patentgrep.com/patent/US5352769A

## CPC

- C07K 14/62: Insulins
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

This disclosure relates to new compounds and to a process for the controlled conversion of such compounds to produce a Protein that comprises an amino acid sequence which is useful as a precursor to human insulin or to a modified human insulin, the amino terminus portion of which sequence does not define a cathepsin C dipeptide removal stop point. The compounds have the formula Met-Y-Protein in which Y is Tyr or Arg.

## Claims

### Claim 1

A compound of the formula Met-Y-Protein in which Protein comprises three amino acid sequence units designated B, A, and X, in which B represents the B-chain of human insulin or a modified B-chain having one or more of the following amino acid modifications:
(1) replacement of the Pro in position 28 by any of the following: Asp, Val, Leu, Ile, Nle, Arg, His, Lys, Phe, Ala, Gly, citrulline, and ornithine;
(2) replacement of the Lys in position 29 by any of the following: L-Pro, D-Pro, D-hydroxyproline, L-hydroxyproline, L-(N-methyl)Lys, D-Lys, L-(N-methyl)Arg, and D-Arg;
(3) replacement of the His in position 10 by Asp;
(4) deletion of the Thr in position 30; A represents the A-chain of human insulin or a modified A-chain, in which the asparagine in position 21 is replaced by alanine; and X represents a moiety joined to the insulin A-chain or modified A-chain at the amino terminus of the A-chain and to the insulin B-chain or modified B-chain at the carboxy terminus of the B-chain, the amino terminus portion of which sequence does not define a cathepsin C dipeptide removal stop point, and Y is Tyr or Arg.

### Claim 2 (depends on 1)

Compound of claim 1, in which Y is Tyr.

### Claim 3 (depends on 1)

Compound of claim 1, in which Y is Arg.

### Claim 4 (depends on 1)

Compound of claim 1, in which A is the A-chain of human insulin.

### Claim 5 (depends on 1)

Compound of claim 1, in which B is the B-chain of human insulin.

### Claim 6 (depends on 1)

Compound of claim 1, in which X is the natural connecting peptide of human proinsulin.

### Claim 7 (depends on 1)

Compound of claim 1, having a modified B-chain in which the Pro in position 28 has been replaced by Lys.

### Claim 8 (depends on 1)

Compound of claim 1, in which the Lys in position 29 has been replaced by Pro.

### Claim 9 (depends on 1)

Compound of claim 1, having a modified B-chain in which the Lys in position 29 has been replaced by Pro.

### Claim 10

A compound of the formula
Met-Y-Protein
in which Y is Arg and Protein is human proinsulin in which Pro at position 28 of the B-chain is replaced with Lys and in which Lys at position 29 of the B-chain is replaced with Pro.