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Production of thiolsubtilisin YaB and its application to peptide-ligation

硫醇枯草杆菌蛋白酶YaB的制备及其在肽连接中的应用

基本信息

批准号：
05556014
负责人：
YAMASAKI Makari
金额：
$ 9.54万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
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

项目摘要

Alkaline elastase produced by alkalophilic Bacillus YaB starin is a serine-protease having high optimum pH and effectively hydolyzes elastin, a sclero-protein. The enzyme has a 55% amino acid sequence homology to subtilisin BPN, and can be called as subtilisin YaB.The substitution of Ser221 at the catalytic center of subtilsin BPN, to Cys causes loss of protease activity but the substituted one, called as thiolsubtilisin, still retains peptide-ligase activity. So the thiolsubtilisin is greatly interested as a new tool for biotechnology. The production of thiolsubtilisin by a recombinant Bacillus subtilis is, however, highly difficult so far. Subtilisins are synthesized in vivo as precursors having pro-sequences. The pro-sequences are auto-catalytically processed and known as essential guide sequences for active conformation. The aim of this study is to try to produce thiolsubtilisin YaB in Bacillus subtilis by a new genetic engineering method. We succeeded to produce active subtilsin YaB in Bacillus subtilis which carried a plasmid encoding two separate genes for pre-pro and pre-mature polypeptides of subtilsin YaB,though the efficiency is low. The production of thiolsubtilisin YaB in a similar fashion is, however, not successful. In the next place, we aimed to secrete pro-thiolsubtilisin YaB by Bacillus subtilis WB600 strain(6 extracellular proteases-negative mutant) carrying a plasmid encoding pre-pro-mature polypeptide of thiolsubtilisin YaB.We observed a secreted 52 kDa species positive in the western blotting. The proteins was proved to be processed to a mature size species (26kDa). This result clearly shows that the 52 kDa species is the pro-form and that the 26 kDa pro-form is 36 kDa. The observed molecular mass of 52 kDa, therefore, suggests an unusual structure of the pro-form.

由嗜碱芽孢杆菌YaB starin产生的碱性弹性蛋白酶是一种丝氨酸蛋白酶，具有高的最适pH，并有效地水解弹性蛋白，一种硬蛋白。该酶与枯草杆菌蛋白酶BPN具有55%的氨基酸序列同源性，并且可以被称为枯草杆菌蛋白酶YaB。枯草杆菌蛋白酶BPN的催化中心的Ser 221被Cys取代导致蛋白酶活性的丧失，但是被取代的枯草杆菌蛋白酶（thiolsubtilisin）仍然保留肽连接酶活性。因此，硫代枯草杆菌蛋白酶作为一种新的生物技术工具，引起了人们极大的兴趣。然而，迄今为止，通过重组枯草芽孢杆菌生产硫代枯草杆菌蛋白酶是非常困难的。枯草杆菌蛋白酶在体内合成为具有前序列的前体。前序列是自催化加工的，并且被称为活性构象的必需指导序列。本研究的目的是尝试用一种新的基因工程方法在枯草芽孢杆菌中生产硫代枯草杆菌蛋白酶YaB。我们成功地在枯草杆菌中产生了具有活性的枯草杆菌蛋白酶YaB，该枯草杆菌蛋白酶YaB携带编码枯草杆菌蛋白酶YaB的前原和前成熟多肽的两个独立基因的质粒，但效率较低。然而，以类似的方式生产硫代枯草杆菌蛋白酶YaB并不成功。其次，我们利用枯草芽孢杆菌WB600菌株（6种胞外蛋白酶阴性突变株），通过构建编码硫代枯草杆菌蛋白酶YaB前原成熟多肽的质粒，分泌硫代枯草杆菌蛋白酶YaB前体。蛋白质被证明加工成成熟大小的种类（26kDa）。该结果清楚地表明，52 kDa物质是前体形式，26 kDa前体形式是36 kDa。因此，观察到的分子量为52 kDa，表明前体形式的不寻常结构。