Preparation of unglycosylated human caseinomacropeptide by engineering DAB Escherichia coli.
Human caseinomacropeptide (hCMP) is 65 amino acids in length and was originally derived from the C terminus of human milk kappa-casein. As it is highly abundant in both essential amino acids and branched amino acids, it could be developed as a practical food and even as medicinal nutrition for patients. This study was undertaken to prepare recombinant hCMP without glycosylation using recombinant plasmid and prokaryotic expression system. The gene encoding hCMP was chemically synthesized and directly inserted into the pET28a(+) vector and then expressed in Escherichia coli BL21(DE3). The maximum amount of soluble protein was obtained by incubation with 0.5 mM isopropyl-alpha-D-thiogalactopyranoside at 30 degrees C for 4 h and accounted for 40% of the total intracellular protein. Most of the expressed fusion proteins, located in the cell periplasm and cytoplasm, could be adsorbed by nickel affinity chromatography and eluted with buffer containing 300 mM imidazole. The fusion proteins were cleaved by enterokinase to remove the 6-His tag. Gel filtration chromatography with Sephadex G-10 was performed for desalting and purification. A final yield of 25 mg of the mature protein with high purity up to 99% was obtained from 1 L of E. coli culture. The purified protein was confirmed by MALDI-TOF-MS analysis. This study overcame the problem of glycosylation in hCMP and established a novel approach for the preparation of unglycosylated hCMP.
Liu
Fangjie
F
College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, People's Republic of China.
Liao
Liang
L
Chen
Jinchun
J
D002364:Caseins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D002364:Caseins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C065989:caseinomacropeptide; D014357:Trypsin