Peptide synthesis by enzymatic method, especially its application for total synthesis of human insulin

酶法合成肽，特别是在人胰岛素全合成中的应用

• 批准号: 05806014
• 负责人: MORIHARA Kazuyuki
• 金额: $ 0.32万
• 依托单位: university of East Asoa Gramt-in-Aid for Scientific Research (C)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05806014/
• 关键词: V8 protease; trypsin; total synthesis of insulin; lysyl endopeptidase; fragment condensation; single chain insulin; インスリン; ペプチド合成; ぶたインスリン; ひとインスリンの全合成

For the purpose of total synthesis of human insulin, enzymatic method was applied for the fragment condensation of A or B chain, together with the preparation of single chain insulin by coupling of fragments A and B chains.Eight insulin fragments were synthesized using a peptide synthesizer (Shimadzu model PSSM-8). For coupling, Staphylococcus aureus V8 protease (abbrev.SAP), trypsin (abbrev.T) and lysyl endopeptidase (abbrev.L) were used. The reaction was performed in the persence of high concentration of organic cosolvent (40% dimethylformamide). The reaction mixture contained 2 mM amine component and 10 mM carboxyl component, pH 6-7, which was kept for 1-3 days at room temperature. The other experimental conditions are described below. The coupling yield was determined using high performance liquid chromatography (HPLC). The result indicated that the peak of amine component disappeared and a new peak appeared in HPLC in all the coupling reactions below. Amino acid analysis of the respective new peaks in HPLC indicated that the new peaks were the coupling products as had been expected.Abbrev. : A ; insulin A chain, Cys6-Cys11 (S-S bridge), Cys7-Acm, Cys20-Acm.B ; insulin B chain, Cys7-Acm, Cys19-Acm.Acm, acetoamidemethy1..The numbers in parentheses are the amino acid ones in the respective sequences.

为了实现人胰岛素的全合成，采用酶促法对A链或B链进行片段缩合，并将A链和B链片段偶联制备单链胰岛素。采用肽合成仪（Shimadzu模型PSSM-8）合成8个胰岛素片段。对于偶联，金黄色葡萄球菌V8蛋白酶(简称。采用SAP、胰蛋白酶（缩写为t）和赖氨酸内肽酶（缩写为l）。该反应是在高浓度有机助溶剂（40%二甲基甲酰胺）存在下进行的。反应混合物中含有2 mM胺组分和10 mM羧基组分，pH 6-7，室温保存1-3天。其他实验条件描述如下。采用高效液相色谱法测定偶联率。结果表明，在所有的偶联反应中，胺组分的HPLC峰消失，出现了一个新的峰。对新峰的氨基酸分析表明，新峰是偶联产物，符合预期。： a；胰岛素A链，Cys6-Cys11 （S-S桥），Cys7-Acm, Cys20-Acm。B;胰岛素B链，Cys7-Acm, Cys19-AcmAcm, acetoamidemethy1 . .括号内的数字是各自序列中的氨基酸。