Preparation Of Unglycosylated Human Caseinomacropeptide By Engineering Escherichia Coli

Human caseinomacropeptide (hCMP) is64amino acids in length and was originally derived from the C-terminus of human milk K-casine. As it is highly abundant in essential amino acids and branched amino acids, it could be developed as a practical food and even as medicinal nutrition for patients. The recombinant hCMP without glycosylation was prepared using recombinant plasmid and prokaryotic expression system.The hCMP gene was chemically synthesized based on the amino acid sequence of hCMP by using the preferred codons of E. coli. Upstream of the synthesized gene was a BamH I site for ligation and downstream of the gene a SacI site was located for the direction of ligation during the construction of the expression vector. The hCMP gene was inserted directly into pET-28a(+) by both BamHI and SacI sites to generate the pET-28a(+)-hCMP construct which was transformed into E. coli DH5a strain for sequencing. Finally, the plasmid pET-28a(+)-hCMP was transformed to E. coli BL21(DE3) for the expression of hCMP.To get high level expression of recombinant hCMP, the optimal process was studied in detail. The optimal experiment conditions for the genetic engineered E.coli were as follows:E. coli BL21(DE3) were incubated in2×YT media containing100mg·L-1kan at37℃. When the optical density at600nm reached0.6-0.8, IPTG was added at0.2mM·L-1final concentration, and the bacteria were incubated for8h, the yield of hCMP reached the highest. It was about62.8%of the totle bacteria protein based on SDS-PAGE analysisMost of the target proteins were located in the cell periplasmic space and cytoplasm, while on the other hand, the target peptide located in the inclusion body was at a low relative level. The target proteins could be adsorbed by nickel affinity chromatography and eluted with buffer containing300mM/L imidazole. The fusion proteins were cleaved by enterokinase to remove6-His tag. Gel filtration chromatography with Sephadex G-10was performed for desalting and purification. A final yield of25mg of the mature protein with high purity up to97%was obtained from one liter of E. coli culture. The recombinant hCMP was further confirmed by Western blotting using anti-His6tag monoclonal antibody and MALDI-TOF-MS analysis.This study overcame the problem of glycosylation in hCMP and established a novel approach for the preparation of unglycosylated hCMP.