文献类型：期刊文献

中文题名：胰岛素原C肽多聚体基因在大肠杆菌中的表达及重组C肽在水溶液中的稳定性(英文)

英文题名：Expression of C-peptide Multiple Gene Copies in Escherichia coli and Stabilities of C-peptide in Aqueous Solution

作者：李素霞[1];田丽萍[2];刘海峰[1];张育坚[2];胡晓波[2];龚毅[2];袁勤生[1]

机构：[1]华东理工大学生物反应器国家重点实验室,上海200237;[2]中国科学院上海生命科学研究院生物工程研究中心,上海200233

年份：2003

卷号：35

期号：11

起止页码：986

中文期刊名：生物化学与生物物理学报

收录：CSTPCD;;Scopus;北大核心:【北大核心2000】；CSCD:【CSCD2011_2012】；PubMed;

语种：中文

中文关键词：胰岛素原C肽;RP-HPLC;多聚体基因;大肠杆菌;表达;水溶液

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

摘要：合成了胰岛素原C肽三聚体的基因 ,并在大肠杆菌中得到高效表达。表达的融合蛋白质通过设计的特异性位点的酶切得到重组的人胰岛素原C肽单体。融合蛋白质以可溶性蛋白质的形式表达 ,表达量约为 80mg/L。Ni NTA亲和柱可有效地从细胞裂解的上清液中分离纯化融合蛋白质 ,得到纯度大于 70 %的融合蛋白质 37.5mg/L。融合蛋白质可经胰蛋白酶 /羧肽酶B双酶切有效释放天然的C肽。释放的C肽经氨基酸组成分析、与标准对照的RP HPLC分析和免疫发光法定量证实与天然人C肽完全相同。C肽经RP HPLC纯化后可得 ,总的收率为 1 .5mg/L ,纯度大于 95 %。考察了C肽在冻干过程中及在水溶液中的稳定性。结果表明C肽在冻干过程中稳定 ,在水溶液中其稳定性受 pH和温度的影响。在 pH 3和pH 7.4缓冲液中 ,37℃或 70℃下 ,C肽的降解符合一级动力学。C肽冻干品直接溶解于pH 9的缓冲液中 ,立即降解 ,降解产物在 37℃和 70℃下基本稳定。C肽冻干品直接溶解于pH 3的缓冲液中 ,立即发生降解反应 ,随后可观察到随温度升高和时间延长 ,降解反应的进行 ,37℃、1 0h ,可观察到约 80 .3%的C肽保持 ,而在 70℃、1 0h ,只有 4 3%C肽保持。C肽在pH 7.4最稳定 ,37℃、6h或 1 0g/LBSA存在下 ,70℃、3h ,未观察到降解产物。PH 7.4、37℃、1 0h ,在有?
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 ℃ or 70 ℃, only 40.3% of C peptide was remained after 10 h at 70 ℃. The maximum stability was achieved at pH 7.4, more than 90% of C peptide were detected at pH 7.4 and 37 ℃ after 10 h and at pH 7.4 and 70 ℃ after 5 h. 99% and 96% of C peptide was remained at pH 7.4 and 37 ℃ after 10 h with and without 10 g/L BSA respectively.