文献类型：期刊文献

英文题名：Expression of C-peptide multiple gene copies in Escherichia coli and stabilities of C-peptide in aqueous solution

作者：Li, SX; Tian, LP; Liu, HF; Zhang, YJ; Hu, XB; Gong, Y; Yuan, QS

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Chinese Acad Sci, Shanghai Inst Biol Sci, Res Ctr Biotechnol, Shanghai 200233, Peoples R China

年份：2003

卷号：35

期号：11

起止页码：986

外文期刊名：ACTA BIOCHIMICA ET BIOPHYSICA SINICA

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000186961400004)】；

语种：英文

外文关键词：proinsulin C-peptide; multiple copies; expression; stability; RP-HPLC

摘要：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 IMN41JLITE 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 degreesC or 70 degreesC, only 40.3% of C-peptide was remained after 10 h at 70 degreesC. The maximum stability was achieved at pH 7.4, more than 90% of C-peptide were detected at pH 7.4 and 37 degreesC after 10 h and at pH 7.4 and 70 degreesC after 5 h. 99% and 96% of C-peptide was remained at pH 7.4 and 37 degreesC after 10 h with and without 10 g/L BSA respectively.