Design and cloning of the gene for a novel insulin analogue,(B30-homoserine) human insulin

Abstract

In order to prepare a novel human insulin analogue substituted with homoserine at B30 position, (B30-homoserine) human insulin, a synthetic gene was designed by linking directly a gene for B chain with that for A chain. This gene was constructed by enzymatic joining of 10 different synthetic oligonucleotides, and then inserted at the polylinker region of pUC19 plasmid. To achieve a high level of gene expression, the gene fusion technique was employed using amino terminal regions of lacZ gene up to ClaI or Hpal, and either of them has been located under tac promoter. The chemical induction of these fused genes by isopropyl-β-D-thiogalactopyranoside (IPTG) gave a satisfactory level of expression in Escherichia coli harboring the constructed plasmids. It was observed that the fused gene product as a single chain insulin precursor was produced more than 30% of total cell protein of E. coli as a form of inclusion body.