Production of thiolsubtilisin YaB and its application to peptide-ligation

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，具有较高的最适pH，并能有效地水解硬蛋白弹性蛋白。该酶的氨基酸序列与枯草杆菌毒素BPn的同源性为55%，可称为枯草杆菌YaB。枯草杆菌BPn催化中心的Ser221被Cys取代后失去了蛋白水解酶活性，但被取代的硫代枯草杆菌蛋白仍保持了肽连接酶的活性。因此，硫代枯草杆菌蛋白作为一种新的生物技术工具受到了极大的关注。然而，到目前为止，利用重组枯草芽孢杆菌生产硫代枯草杆菌是非常困难的。枯草杆菌毒素是在体内合成的，作为具有前序列的前体。前序列是自动催化处理的，被称为活性构象的基本指导序列。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菌素Yab。我们在枯草芽孢杆菌中成功地产生了活性枯草杆菌Yab，它携带了编码枯草杆菌Yab前前体和早熟多肽的两个独立基因，但效率较低。然而，以类似方式生产硫代枯草杆菌YAB的做法并不成功。其次，我们的目标是在枯草芽孢杆菌WB600菌株(6个胞外酶阴性突变体)中分泌前硫代枯草杆菌YAB，该菌株携带编码硫代枯草杆菌YAB的早熟多肽。经鉴定，该蛋白已加工成26 kDa的成熟蛋白。这一结果清楚地表明，52 kDa的物种是亲型，26 kDa的亲型是36 kDa。因此，观察到的52 kDa的分子质量表明了前体的一种不寻常的结构。

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.