非纯培养物细菌蛋白酶DNA片段的克隆与表达

为了构建更多的蛋白酶基因工程菌,以及进行蛋白酶基因的直接进化研究,从非纯培养细菌总DNA中扩增各种编码蛋白酶的DNA片段.根据MEROPS和GenBank数据库中的枯草杆菌类蛋白酶的编码区和成熟肽编码序列设计并合成了10条引物.富集培养胞外蛋白酶产生菌并提取了12个总DNA样品,分别用每对引物在降落PCR (TouchdownPCR, TD-PCR)条件下进行蛋白酶编码序列的扩增.选择了19个长800 ~1 200 bp的扩增片段测序,其结果为: 8个是蛋白酶DNA片段,它们应属于4种不同的蛋白酶基因序列;同一对引物扩增到的基因序列差异性可达到32%,说明只使用基于已知序列的PCR方法从混合菌中获得新蛋白酶基因是可行的.将克隆到的1个与碱性蛋白酶E (GenBank No.AJ539133)的编码区99%相似的蛋白酶DNA片段插入pTWIN1载体,在大肠杆菌ER2566中进行表达.结果表明,表达的成熟蛋白酶可分泌到培养基中,能在牛奶平板上产生水解圈,对大肠杆菌有致死作用.图5表3参14

Cloning and Expression of Bacterial Peptidase-coding DNA Fragments from Impure Culture

In order to construct a large number of recombinant strains producing peptidases, as well as to make directed evolution of peptidase genes, cloning of peptidase-coding DNA fragments from impure culture was carried out. Ten primers were designed and synthesized. Bacteria producing extracellular peptidases were enriched for total DNAs. Touchdown PCR (TD-PCR) was done and among the products, 19 fragments with 800~1 000 bp in length were selected for sequence analysis. Eight of them were found as peptidase-coding DNAs of 4 genes named as NK2-SU1, AprE-SU1, SUBJ-SU1 and KPR-SU1. Difference in nucleotide sequences among these genes amplified with the same pair of primers reached 32%. One fragment that was 99% similar to the coding sequence of alkaline protease E (GenBank No., AJ539133) was inserted into the expression vector pTWIN1 of Escherichia coli ER2566. The active product secreted into medium produced a hydrolyzed zone on the defatted milk plate and was lethal to E. coli. Fig 5, Tab 3, Ref 14