Synthesis of Biologically Active Polypeptides Mediated by Highly Specific Serratia 56K Protease

肽合成是由蛋白质介导的，它的反应发生在没有裂解条件和少量副产品的情况下。Serratia Protease （SP） has high substrate specificity in hydrolysis of a peptide bond at the carboxyl side of Arg residue。我们发现了一个SP催化剂，它形成了一种具有高特异性的烷基肽键。在四肽合成模型中，Abz-Gly-Phe-Arg-Xaa-Nba（Xaa ： Leu， Ala， Gly， Abz ：2-氨基苯唑醇， Nba ：4-硝基胺）， SP催化了以下顺序中的烷基键形成， Leu > Ala， Gly，指示SP在肽键形成中具有高顺序选择性。为了将蛋白质应用于大肽的合成，我们试图合成一个模型肽Bz-Gly-Arg-Gly-Phe-Arg-Leu-NH_2包含特定的Arg-Leu和非特定的Arg-Gly债券。However，该产品可能无法被发现在Arg-Leu粘结形成过程中的Arg-Gly粘结剂的水解作用。这一结果取决于 ... More 通过使用任何种类的特定蛋白质来合成肽键的水合作用，开发一种有效的方法，以通过使用蛋白质来合成生物活性的肽，它必须找到一个溶剂系统，其中的溶剂化基础包含部分或完全受保护的肽，由方便的肽合成方法和那些保持蛋白质活性在高年级的产品中。为了这一目的，我们研究了肽系统模型中的水-溶解有机溶剂和水的各种混合物，Abz-Gly-Phe-Arg-Leu-Nba，由trypsin介导。在一个4%H_2O/HFIP/DMF的溶剂系统中，酶催化了83%的yield的反应，而50%H_2O/DMF的反应则经常被其他研究人员使用。它指出，HFIP是一种良好的溶剂具有高性能以溶解性的肽基底，以及含有低水的溶剂转移，使肽键形成具有较高的水平。我们将该溶剂系统应用于人类生长激素释放因子的综合体（hGRF （1-29）-NH_2）。受保护的肽片段是由固体相方法合成的。这些片段是由trypsin的援助所限定的解决方案方法提供两个大的片段。这一步的年薪估计几乎是90%。部分受保护的hGRF （1-29）-NH_2被RP-HPLC保护和净化以给予hGRF （1 -29）-NH_2，其中RP-HPLC和tryptic hydrolization中的肽映射被SP介导的结合、序列特定合成方法是不成功的，但由trypsin的药物开发的生物活性肽合成方法的有效方法。Less（低）

Peptide synthesis mediated by proteases has an advantage that reactions proceed under mild condition without racemization and with few side products. Serratia Protease (SP) has high substrate specificity in hydrolysis of a peptide bond at the carboxyl side of Arg residue. We have found that SP catalyzes the formation of arginyl peptide bond with high specificity. In a model tetrapeptide synthesis, Abz-Gly-Phe-Arg-Xaa-Nba (Xaa : Leu, Ala, Gly, Abz : 2-aminobenzoyl, Nba : 4-nitrobenzylamine), SP catalyzed the arginyl bond formation in the following order, Leu >> Ala, Gly, indicating that SP has high sequence selectivity in the peptide bond formation. To apply the protease to the synthesis of large peptides, we attempted to synthesize a model peptide Bz-Gly-Arg-Gly-Phe-Arg-Leu-NH_2 which contains the specific Arg-Leu and non-specific Arg-Gly bonds. However, the product could not obtained due to hydrolysis of the Arg-Gly bond during formation of the Arg-Leu bond. This result indicated that … More hydrolysis of a peptide bond can not be avoided by use of any kind of specific protease.To develop an efficient method to synthesize biologically active peptides by the use of proteases, it is necessary to find a solvent system which solubilizes substrates containing partially or fully protected peptides prepared by the convenient peptide-synthetic method and which keeps proteases active to give products in high yields. For this purpose, we examined a various mixtures of water-miscible organic solvents and water in the model peptide system, Abz-Gly-PheーArg-Leu-Nba, mediated by trypsin. In a solvent system of 4% H_2O/HFIP/DMF, the enzyme catalyzed the reaction in an 83% yield, while in a 30% yield in 50% H_2O/DMF which was frequently used by other researchers. It is noted that HFIP is a good solvent having high ability to solubilizes peptide-substrates and that the low-water-containing solvent shifts the reaction equilibrium to the peptide-bond formation to give a high yield.We applied this solvent system to the synthesis of the active fragment of human growth hormone releasing factor (hGRF (1-29) -NH_2). The protected peptide fragments were synthesized by the solid-phase method using the oxime resin. These fragments were coupled by the solution method to give two large segments which were condensed by the aid of trypsin. The yield in this step was estimated as nearly 90%. The partially protected hGRF (1-29) -NH_2 was deprotected and purified by RP-HPLC to give hGRF (1-29) -NH_2, which was identified by RP-HPLC and peptide mapping of the tryptic hydrolyzates.In conclusion, sequence-specific synthesis of peptides mediated by SP was not successful, but efficient method for synthesis of a biologically active peptide by the aid of trypsin has been developed. Less