Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Insulin; related peptides
Patent
1990-12-13
1993-04-13
Cashinon, Jr., Merrell C.
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Insulin; related peptides
514 3, 514 4, A61K 3702, A61K 3726, C07K 500
Patent
active
052024156
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to novel propeptides More specifically, the invention relates to novel insulin precursors which can be used in the preparation of human insulin or insulins showing inherent protracted action or accelerated action. Moreover, the invention relates to DNA sequences coding for said insulin precursors as well as a process for the preparation of such precursors and to a process for the preparation of human insulin or insulin analogues.
BACKGROUND ART
In severe or chronic cases the disease of Diabetes is usually treated with injection preparations containing insulin, e.g. porcine insulin, bovine insulin or human insulin.
A number of different processes for the biosynthetic production of human insulin are known. Common to all of them is that the DNA strand coding for either the entire proinsulin, a modified form hereof or for the A and B chain separately is inserted into a replicable plasmid containing a suitable promoter. By transforming this system into a given host organism a product can be produced which can be converted into authentic human insulin in a manner known per se, cf. e.g. EP B1 85,083 or EP B1 88,117.
Some known processes for biosynthesis of proinsulin or similar insulin precursors and there conversion into insulin are described below.
Proinsulin may be prepared biosynthetically by using the method disclosed in the specification of European patent application No. 121,884. In this method the gene coding for proinsulin is inserted into a yeast strain and after culturing such transformed yeast strain proinsulin can be isolated from the culture medium. Hereafter, proinsulin can be converted into insulin in a manner known per se. Yields of proinsulin obtained by this method are, however, unsatisfactory low for commercial production.
Insulin precursors of the formula B-X-A wherein B and A represent the B and A chain, respectively, of human insulin and X represents a polypeptide comprising at least 2 amino acid residues, preferably from 6 to 35 amino acid residues, are known from the specification of Danish patent application No. 5284/87. The precursors can be enzymatically digested into human insulin by treatment with trypsin and carboxypeptidase B in the presence of certain metal ions.
European patent application No. 195,691 discloses closely related insulin precursors of the formula B-X-Y-A wherein B and A represent the B and A chain, respectively, of human insulin, cross-linked through sulphur bridges as in human insulin, and X and Y each represents a lysine or arginine residue, as well as the preparation of said precursors. These precursors can be enzymatically digested into human insulin by treatment with trypsin and carboxypeptidase B. Moreover, said precursors can undergo tryptic digestion into des-B30-insulin; however, a considerable amount of A.sub.o Arg-des(B30)-insulin is formed which only slowly undergoes further digestion.
It is an object of the invention to provide novel insulin precursors which are generated in high yields in yeast and which furthermore can be converted into human insulin or insulin analogues with minimal formation of undesired by-products.
The insulin precursors of the invention are characterized by the following amino acid sequence human insulin starting from the N-terminus, A(1-21) are the 21 amino acid residues of the A chain of human insulin, X.sub.1 represents a peptide bond or one or more arbitrary amino acid residues, X.sub.2 represents Glu or Asp, and Y.sub.1 and Y.sub.2 each represents Lys or Arg, the positions A6 and A11, A7 and B7, and A20 and B19, respectively, are connected through sulphur bridges, and, if desired, one or more of the amino acid residues of the chains B(1-29) and A(1-21) are substituted by another amino acid residue.
The invention is based on the surprising recognition that the above insulin precursors are either generated in an extremely high yield as compared with the compound B-Lys-Arg-A known from European patent application No. 121,884 or that when these precursors are digested by trypsin
REFERENCES:
patent: 4430266 (1984-02-01), Frank
Dayhoff et al. Atlas of Protein Sequence and Structure, vol. 5 (1972) 89-99.
Rudinger, Peptide Hormones (Ja Parsons ed 1976) 1-6.
Clausen Ib G.
Jensen Einer B.
Jonassen Ib
Svendsen Allan
Cashinon, Jr. Merrell C.
Celsa B.
Novo Nordisk A S
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