Expression of human insulin-like growth factor Ⅱ as a fusion protein in E. coli, purification of the protein, and preparation of monoclonal antibody to the protein

Abstract

The plasmid containing human insulin-like growth factor-II(IGF-II) cDNA was modified to delete nontranslated region and portions of coding region for IGF-II at both 5``-and 3``-end of the cDNA sequence. A gene encoding mature IGF-II was prepared from the modified cDNA sequence and double-stranded synthetic linkers. The synthetic linkers was designed to resupply the missing coding information for mature IGF-II polypeptede. For the expression of mature IGF-II in E. coli, the expression vector, pSIG was constructed by placing the gene encoding mature IGF-II, in correct frame, just after translational initiation codon, ATG, of pASI. In the pSIG, the IGF-II gene was located under the control of lamda phage $P_L$ promoter. The E. coli strain M5248 harbouring pSIG was induced to express human IGF-II polypeptide by rapid shifting of temperature ($32^\circ\,\rightarrow\,42^\circ$C). However, recombinant IGF-II was not detectable by Commassie Blue staining of the total cellular lysates which were subjected to SDS-polyacrylamide gel electrophoresis. In order to improve expression of IGF-II gene in E. coli, we decided to fuse the gene to 3``-end of lac Z gene. We constructed another expression plasmid, pYRM-$II_4$ by inserting the coding sequence for Asp-Pro-Met-IGF-II into ClaI site within lacz gene of pCTIO. Thus, the fused lacZ``-IGF-II gene was located under the control of tac promoter of pCTIO. The mature IGF-II have been overgproduced as C-terminal part of $\beta$-galactosidase composed of N-terminal 280 amino acids. The E. coli JM109 cells harbouring pYRM-$II_4$ was induced to express the fused gene by 2 mM IPTG at early log phase. The level of expression of IGF-II was observed 6 hours after induction. It was estimated by densitometric scanning that the recombinant fusion protein accounted about 25\% of the total cellular proteins. The IGF-II fusion protein formed inclusion bodies, more and less homogenous protein aggregates, inside the cells as shown by transmissio...