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.

为了完全合成人胰岛素，将酶促方法应用于A或B链的片段凝结，以及通过碎片A和B链的偶联来制备单链胰岛素。 （Shimadzu型号PSSM-8）。为了耦合，使用了金黄色葡萄球菌V8蛋白酶（Abbrev.sap），胰蛋白酶（Abbrev.t）和赖氨酸内肽酶（Abbrev.l）。反应是在高浓度的有机溶剂上（40％二甲基甲酰胺）的持久性进行的。反应混合物包含2 mM胺分量和10 mM羧基成分PH 6-7，在室温下保存1-3天。下面描述了其他实验条件。使用高性能液相色谱（HPLC）确定耦合产率。结果表明，在下面的所有耦合反应中，胺分量的峰消失了，HPLC中出现了一个新峰。对HPLC中各个新峰的氨基酸分析表明，新峰是偶联产物，如预期的。 ：a;胰岛素A链，Cys6-Cys11（S-S桥），CYS7-ACM，CYS20-ACM.B；胰岛素B链，Cys7-ACM，Cys19-Acm.acm，乙酰胺甲基1.括号中的数字是相应序列中的氨基酸。