Molecular genetic study on activation, processing and secretion of Pseudomonas aeruginosa alkaline protease

1)铜绿假单胞菌碱性蛋白的定点突变对碱性蛋白的活性部位和钙结合部位进行定点突变。PAPE1(APRA，PA的结构基因，插入到pUC18中)，获得了与突变位点有关的Kpn1-Pst1片段(1.3Kb)。将该片段插入到载体M13mp18中，形成单链DNA，按照Kunkel的方法进行定点突变。构建了锌配体(H176*L)、催化位(E177*Q)和钙结合位(D356*A、D365*A)突变体。通过DNA测序证实了突变的存在。2)pWE分泌物的构建用聚合酶链式反应的方法构建分泌物的分泌物(apr D/E/F)，但构建失败。PJUEK72(8.8kb)，apr D/E/F/A(PA可分泌)pAGS7(7.0kb)，apr D/E/F/F(仅有分泌机械)3)PA定点突变对其在大肠杆菌中分泌的影响。用含有1%脱脂牛奶和LB-培养基的琼脂平板法检测PA的分泌。结果表明，在携带pJUEK72或pAGS7+pAPE1的大肠杆菌中观察到了晕环的形成，而在携带突变基因的菌株中没有观察到晕环的形成。用培养滤液进行聚丙烯酰胺凝胶电泳和Western blotting分析，发现突变基因在活性部位的产物比PA大，但在钙结合部位有小分子水解物。并对结果进行了讨论。

1) Site-directed mutagenesis of Pseudomonas aeruginosa alkaline proteaseTo perform the site-directed mutagenesis at the active site and Ca-binding site of the alkaline protease (abbrev. PA), the Kpn1-Pst1 fragment (1.3Kb), involving with the site to be mutated, was prepared from plasmid pAPE1 (aprA,structural gene of PA,inserted in pUC18). The fragment was inserted in vector M13mp18, resulted in the formation of single strand DNA.The site-directed mutagenesis was made according to the method of Kunkel. The following mutant was constructed ; Zn-ligand (H176*L), catalytic site (E177*Q), and Ca-binding site (D356*A,D365*A). The mutation was confirmed by DNA-sequencing.2) Construction of secretion machienery of PAWe tried to construct the secretion machienery (apr D/E/F) using PCR method, but failed. So, the following two plasmids were supplied by the courtesy of Dr.Murgier of CNRS in France.pJUEK72 (8.8KB) ; apr D/E/F/A (PA can be secreted)pAGS7 (7.0Kb) ; apr D/E/F (only secretion machinery)3) Effect of site-directed mutagenesis of PA for its secretion in Escherichia coliThe transfection was done in E.coliC-600 or JM 109 with the plasmid (pAGS7+pAPE1 or pAGS7+muteted pAPE1) to investigate the effecton seceretion. The secretion of PA was examined by the formation of halo using with the agar-plate assay containing LB-medium and 1% skim milk. The result indicated that halo formation was observed in E.coli carryning the plasmid of pJUEK72 or pAGS7+pAPE1, but not in the strains carrying the mutated genes. The cultural filtrate was used for the study of polyacrylamide gel electrophoresis and Western blotting, which indicated that a little larger product than PA (50 KDa) was obtained in the mutated gene at the active site, but small molecular hydrolyzates was observed in the mutated genes at Ca-binding site. The discussion was made on the results.