Preparation of Unglycosylated Human Caseinomacropeptide by Engineering DAB Escherichia coli

• Published in: Journal of agricultural and food chemistry 2008-02, Vol.56 (3), p.889-893
• Main Authors: Liu, Fangjie, Liao, Liang, Chen, Jinchun
• Format: Article
• Language: English
• Subjects: amino acid composition; Biological and medical sciences; breast milk; Caseins - biosynthesis; Caseins - chemistry; Caseins - genetics; Chemical Aspects of Biotechnology/Molecular Biology; Escherichia coli; Escherichia coli - genetics; expression; Food industries; Food microbiology; food technology; functional foods; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic Vectors - genetics; genetically engineered microorganisms; Glycosylation; Humans; identify; kappa-casein; molecular cloning; novel foods; Peptide Fragments - biosynthesis; Peptide Fragments - chemistry; Peptide Fragments - genetics; Protein Engineering - methods; purification; recombinant DNA; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin - metabolism; Unglycosylated hCMP; unglycosylated human caseinomacropeptide
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf072585n

Human caseinomacropeptide (hCMP) is 65 amino acids in length and was originally derived from the C terminus of human milk κ-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-α-d-thiogalactopyranoside at 30 °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.