Li Su-Xia, Tian Li-Ping, Liu Hai-Feng, Zhang Yu-Jian, Hu Xiao-Bo, Gong Yi, Yuan Qin-Sheng
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2003 Nov;35(11):986-92.
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.
合成了编码三个胰岛素原C肽拷贝的基因片段，并在大肠杆菌中进行表达，通过对所得基因产物进行位点特异性切割产生重组胰岛素原C肽。融合蛋白在细胞质中以高水平（约80 mg/L）作为可溶性产物表达。镍-氮三乙酸亲和层析有效地从上清液中分离出表达的融合蛋白，得到约37.5 mg/L纯度为70%的融合蛋白。用胰蛋白酶和羧肽酶B对融合蛋白进行酶切有效地释放出天然C肽，重组C肽的总产率超过95%，为1.5 mg/L。氨基酸组成的良好一致性，以及在比较反相高效液相色谱分析和免疫发光C肽定量测定中重组C肽与C肽标准品显示的相似性，表明本报告中获得的重组C肽是天然人C肽。还报道了通过反相高效液相色谱对重组人C肽在水溶液中的化学稳定性进行的研究。重组C肽的降解表现出对pH和温度的显著依赖性。C肽在pH 3或pH 9缓冲溶液中立即发生降解反应。在37℃或70℃的pH 3缓冲溶液中，C肽的降解反应遵循一级动力学，在70℃下10小时后仅剩余40.3%的C肽。在pH 7.4时达到最大稳定性，在pH 7.4和37℃下10小时后以及在pH 7.4和70℃下5小时后检测到超过90%的C肽。在有和没有10 g/L牛血清白蛋白的情况下，在pH 7.4和37℃下10小时后分别有99%和96%的C肽保留。
