STUDIES ON THE SECRETION OF HUMAN PROINSULIN AND INSULIN-LIKE GROWTH FACTORS I AND II IN E.COLI

Abstract

Publisher Summary This chapter discusses the secretion of human proinsulin and insulin-like growth factors I and II in E. coli. The aim is to study structure–function relationships in the hormones of the insulin family by examining the properties of engineered recombinant insulin and IGF's. To achieve this, the expression vector pIN-III-OmpA2 is being used. Fragments containing the genes for proinsulin and both the IGFs have been subcloned into pIN-Ill-OmpA2. Site-specific deletion of the noncoding DNA between the signal peptide coding sequence and the inserted gene is currently being carried out using synthetic oligonucleotides. The secretion of a proinsulin analogue containing 5 additional amino acids at the N-terminus coded for by DNA yet to be deleted is being examined. SDS-PAGE of cell fractions has shown that a protein of the correct molecular weight is present in the periplasm of induced cells, and phase-contrast microscopy has shown that morphological changes also occur. The N-terminal sequencing to check that the OmpA signal peptide has been correctly cleared is also described.