Double-C-peptide human proinsulin.

A fusion gene encoding double-C-peptide human proinsulin was constructed by insertion of a DNA fragment encoding human C-peptide into the 5'-terminal C-peptide coding sequence of a synthetic human proinsulin gene with correct reading frame and over-expressed in E. coli. The purified double-C-peptide human proinsulin shows decreased activity in receptor binding and insulin immune assays as compared with human proinsulin. Disulphide bond reconstitution studies demonstrate that there is not much more influence of the protein concentration on the yield of refolded double-C-peptide human proinsulin. The double-C-peptide human proinsulin shows a 1.86-fold human C-peptide immune activity as compared with that of human proinsulin and gives a good yield of the molecule with correct disulphide bonds in reconstitution studies strongly suggesting the existence of very flexible conformation of the C-peptide.