Expression of C-peptide multiple gene copies in Escherichia coli and stabilities of C-peptide in aqueous solution

• Published in: Sheng wu hua hsüeh yü sheng wu wu li hsüeh pao Vol. 35; no. 11; p. 986
• Main Authors: Li, Su-Xia, Tian, Li-Ping, Liu, Hai-Feng, Zhang, Yu-Jian, Hu, Xiao-Bo, Gong, Yi, Yuan, Qin-Sheng
• Format: Journal Article
• Language: English
• Published: China 01.11.2003
• Subjects: Amino Acid Sequence; C-Peptide - chemistry; C-Peptide - genetics; C-Peptide - metabolism; Chromatography, Affinity - methods; Chromatography, High Pressure Liquid - methods; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; Gene Expression; Hydrogen-Ion Concentration; Molecular Sequence Data; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Solutions; Temperature; Time Factors
• ISSN: 0582-9879

A gene fragment encoding three copies of proinsulin C-peptide was synthesized and expressed in E. coli and the recombinant proinsulin C-peptide was produced through site-specific cleavage of the resulting gene products. The fusion protein was expressed at high level, about 80 mg/L, as a soluble product in the cytoplasm. Ni-NTA affinity chromatography efficiently separated the expressed fusion protein from the supernatant, to obtain about 37.5 mg/L of the fusion protein with 70% purity. Enzymatic digestion by trypsin and carboxypeptidase B of the fusion protein efficiently released native C-peptide, the overall yield of recombinant C-peptide at a purity over 95% was 1.5 mg/L. The good agreement of amino acids composition, together with shown similarities of the recombinant C-peptide to C-peptide standard in the comparative RP-HPLC analysis and IMMULITE C-Peptide quantitative assay, suggested that the recombinant C-peptide obtained in this report was the native human C-peptide. The investigation of the chemical stability of recombinant human C-peptide in aqueous solutions by RP-HPLC was also reported. The degradation of the recombinant C-peptide showed a marked dependence on pH and temperature. The degradation reaction of C-peptide occurred immediately in pH 3 or pH 9 buffered solution. The degradation reaction of C-peptide followed first-order kinetics in pH 3 buffered solution at 37 degrees C or 70 degrees C, only 40.3% of C-peptide was remained after 10 h at 70 degrees C. The maximum stability was achieved at pH 7.4, more than 90% of C-peptide were detected at pH 7.4 and 37 degrees C after 10 h and at pH 7.4 and 70 degrees C after 5 h. 99% and 96% of C-peptide was remained at pH 7.4 and 37 degrees C after 10 h with and without 10 g/L BSA respectively.