Proinsulin derivative and process for producing human insulin

#The present invention relates to a human proinsulin derivative and a process for producing human insulin by using an expression vector comprising a DNA encoding the human proinsulin derivative. More particularly, it pertains to a human proinsulin derivative which has a short peptide sequence between A and B chains of insulin, in place of the C-peptide; an expression vector comprising a DNA encoding the human proinsulin derivative; a microorganism transformed with the expression vector; and a process for producing human insulin by culturing the microorganism in a suitable medium.

#Insulin is a polypeptide hormone secreted by the .beta.-cells of pancreas and takes part in regulating the blood sugar level. It consists of two peptide chains, i.e., A and B chains, which are linked by disulfide bridges at their cysteine residues and is produced by a proteolytic processing of proinsulin in pancreatic .beta.-cells(Insulin: Molecular Biology and Pathology, ed. Ashcroft, F. M. & Ashcroft, S. J. H., IRL Press, Oxford, 1992). Commercially, human insulin has been produced either by enzymatic process wherein an alanine residue at the 30th position of the B-chain of porcine insulin is replaced with a threonine residue through a transpeptidation reaction using trypsin(Markussen, J., Proceedings 1st International Symposium `Neue Insuline`, pp 38-44(1982), ed. Petersen, K. G., et al., Freiburger Greaphische Betriebe, Freiburg); or, by a process which uses genetically engineered E. coli(Chance, R. E., et al., Peptides: Synthesis-Structure-Function, pp 721-728(1981), in Proceedings of the Seventh American Peptide Symposium, ed. Rich, D. H. & Gross, E., Pierce Chemical Co., Rockford, Ill., U.S.A.; and Frank, B. H., et al., Peptides: Synthesis-Structure-Function, pp 729-738(1981), in Proceedings of the Seventh American Peptide Symposium, ed. Rich, D. H. & Gross, E., Pierce Chemical Co., Rockford, Ill., U.S.A.) or Saccharomyces cerevisiae(Thim, L., et al., Proc. Natl. Acad. Sci. U.S.A., 83, pp 6766-6770(1986); and Markussen, J., et al., Protein Engineering, 1, pp 205-213(1987)). As the enzymatic process for producing human insulin from porcine insulin is limited by its high cost, recent studies have been focused on processes for producing human insulin by genetic engineering techniques.

#Chance et al. have reported a process for preparing insulin by: producing each of the A and B chains of insulin in the form of a fusion protein by culturing E. coli which carries a vector comprising a DNA encoding the fusion protein; cleaving the fusion protein with cyanogen bromide to obtain the A and B chains; sulfonating the A and B chains to obtain sulfonated chains; reacting the sulfonated B chain with an excess amount of the sulfonated A chain; and then, purifying the resultant to obtain insulin(Chance, R. E., et al., supra). However, this process has drawbacks in that it is cumbersome to operate two fermentation processes and the reaction step of the sulfonated A and B chains gives a low yield of insulin, making the process inherently impractical.

#The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, in which:

#FIG. 1 shows a schematic diagram for preparing plasmids pT7-T1 and pT7-T2;

#FIG. 2 depicts a schematic diagram for preparing plasmids pT1-hPI and pT2-hPI;

#All references cited herein are hereby incorporated in their entirety by reference.

#In accordance with the present invention, there is provided a human proinsulin derivative having formula (I), hereinafter referred to as "miniproinsulin", wherein a peptide, which consists of 12 or Less amino acids and forms a tight .beta.-turn structure, is inserted between A and B chains of human insulin in place of the C-peptide consisting of 35 amino acids:

#B chain-Z-A chain (I)