Fermentation and purification of lacZ-fused single chain insulin precursor for (B30-homoserine) human insulin

Abstract

In order to produce the single chain precursor of a novel human insulin analogue, (B30-homoserine) insulin, the fermentative behaviors ofEscherichia coli JM103 were studied, which harbors pKBA plasmid carrying a hybrid gene in which the gene for a single chain precursor was fused withlacZ gene undertac promoter. The maximal induction of gene expression was achieved when more than 0.05 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) was supplemented to fermentation medium after 4 h cultivation ofE. coli, and followed by longer than 2-h fermentation. The hybrid protein of the single chain insulin precursor was isolated from cytoplasmic inclusion bodies by dissolving in 8M urea solution, and purified through DEAE-Sephacel and Sephadex G-200 column chromatographies with a recovery of 35%. The finally purified hybrid protein showed a single band on sodium dodecyl sulfate-polyacrylamide gel.