Biosynthesis and periplasmic segregation of human proinsulin in Escherichia coli.

Abstract

A plasmid containing human preproinsulin cDNA inserted into the endonuclease Pst I site of the ampicillinase gene of plasmid pBR322 was modified by excision of large portions of the ampicillinase-coding region to produce a variety of gene fusion combinations, many of which generated proteins detectable with antisera to insulin or human C peptide. In one case a perfect hybrid of the NH2-terminal half of the leader sequence of ampicillinase (residues -23 to -12) with the human preproinsulin prepeptide beginning at residue -13 was formed; the result was the synthesis and secretion of human proinsulin into the periplasmic space. We have characterized this protein immunologically and also by labeling it biosynthetically or by iodination followed by immunoprecipitation and automated amino acid sequence analysis. It contains the A and B chain regions of insulin as well as specific human C peptide immunodeterminants and is convertible to an insulin-like component by tryptic digestion. These results demonstrate that human proinsulin can be produced by bacteria and that this biosynthetic approach should prove feasible for the production of adequate amounts of human proinsulin for a variety of clinical studies and human insulin for therapeutic purposes.