Process for enzymatic replacement of the B-30 amino acid in insu

Details Process for enzymatic replacement of the B-30 amino acid in insu Process for enzymatic replacement of the B-30 amino acid in insu

1982-03-23

1987-02-24

Kepplinger, Esther M.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Using a micro-organism to make a protein or polypeptide

435 69, C12P 2104, C12P 2106

Patent

active

046457402

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

1. Field of the invention
The present invention generally relates to a process for enzymatic replacement of the C-terminal amino acid in the B-chain (B-30) of insulins from various species.
It is well known that insulins from different vertebrate species including mammals and humans differ in their primary structure. Since Sanger in 1958 determined the primary structure of bovine insulin the primary structure of insulins from other vertebrate species has been determined.
These results as summarized in the below figure using the porcine insulin as model indicate that amino acid substitutions can occur at many positions within either chain. However, certain structural features are common to all the insulins, e.g. the position of the 3 disulfide bonds, the N-terminal region of the A-chain, the B23-26 sequence in the C-terminal region of the B-chain, etc.
The differences in the primary structure of some common insulins are seen from the below table:


______________________________________ A-chain B-chain 4 8 9 10 3 29 30 ______________________________________ Bovine Glu Ala Ser Val Asn Lys Ala Sheep Glu Ala Gly Val Asn Lys Ala Horse Glu Thr Gly Ileu Asn Lys Ala Sei whale Glu Ala Ser Thr Asn Lys Ala Porcine Glu Thr Ser Ileu Asn Lys Ala Sperm whale Glu Thr Ser Ileu Asn Lys Ala Dog Glu Thr Ser Ileu Asn Lys Ala Human Glu Thr Ser Ileu Asn Lys Thr Rabbit Glu Thr Ser Ileu Asn Lys Ser Rat 1 Asp Thr Ser Ileu Lys Lys Ser Rat 2 Asp Thr Ser Ileu Lys Met Ser ______________________________________
While the invention is described more fully below with relation to the specific conversion of porcine insulin into human insulin, viz. replacement of B-30 alanine by threonine, it will easily be understood that the described method applies equally well to other types of insulin in that e.g. rabbit insulin may also be converted into human insulin, bovine insulin may be converted into B-30 (Thr) bovine insulin, etc.
2. Background of invention, especially with relation to conversion of porcine insulin into human insulin
The idea of converting porcine insulin into human insulin by semi-synthetic procedure has been an attractive problem in the field of insulin chemistry.
As stated above, human insulin differs from porcine insulin by only one amino acid, the C-terminal residue of the B-chain (B-30) being threonine in human and alanine in porcine insulin, respectively. The exchange of alanine to threonine was initially performed chemically and recently enzymatic procedures have been used. Ruttenberg (1972) (Ref. 1) has described the chemical conversion of porcine insulin into human insulin: esterification to insulin-hexamethylester, hydrolysis with trypsin to desoctapeptide insulin (DOI)-pentamethylester, blocking of the amino terminal residues, chemical coupling with a synthetic octapeptide of the corresponding human insulin sequence, acidic deprotection of the amino-groups, and finally alkaline saponifcation of the methyl ester groups. However, nobody has ever been able to produce pure human insulin by this method, since the chemical procedures, and in particular the final alkaline saponification steps seriously damage the insulin molecule and also isoasparagine at the C-terminal residue of the A-chain is formed (Gattner et al. (Ref. 2)). To prevent this effect Obermeier and Geiger (1976) (Ref. 3) have carried out the fragment condensation without protection of the side chain carboxyl groups of DOI. They could isolate human insulin after extensive purification, but only in very low yields. Similar approaches have been taken by Gattner et al. (Ref. 2), using various insulin fragments. However, using the chemical methods nobody has so far prepared pure human insulin in more than trace amounts.
M. Bodanszky et al. provides a process for preparing human insulin in U.S. Pat. No. 3,276,961 wherein human insulin was ostensibly prepared from other animal insulins by an action of an enzyme such as carboxypeptidase A and trypsin in the presence of threonine. This pr

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Johansen et al, Carls. Res. Commun., 41(1): 1-14 (1976).
Chem. Abstracts 94:103810 j Gattner et al. Insulin: Chem. Struct. Funct. Insulin Relat. Horm., Proc. Int. Insulin Symp., 2nd 1979 (Pub. 1980).
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