Biological activity of rat proinsulin synthesized by Escherichia coli

Abstract

Fused proteins possessing insulin antigenic determinants are synthesized and secreted by several strains of Escherichia coli K-12 bearing plasmids containing cDNA copies of rat proinsulin mRNA. Small amounts of the secreted, fused protein from E. coli strains χ1776 and HB101 bearing plasmid p 147 were partially purified and assayed for biological activity by determining stimulation of [1- 14C]glucose oxidation to 14CO 2 in the rat epididymal fat pad assay. Guinea pig anti-insulin serum (GPAIS) was used to suppress glucose oxidation stimulated by insulin. After mild treatment with trypsin, fused protein stimulated 14CO 2 production, and over 90% of this activity was suppressible by GPAIS. Untrypsinized fused protein demonstrated no GPAIS-suppressible 14CO 2 production. Larger amounts of fused protein were obtained by transformation of strain PR 13 of E. coli in which the yield of eukaryotic proteins is increased approx. 50-fold. A single intravenous injection of trypsinized fused protein from E. coli PR13 containing plasmid p287.47 resulted in a rapid decline in the plasma glucose levels of fasted mice, and the hypoglycemic effect persisted for at least 120 min.