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

铜绿假单胞菌碱性蛋白酶激活、加工和分泌的分子遗传学研究

• 批准号: 07660126
• 负责人: MORIHARA Kazuyuki
• 金额: $ 1.34万
• 依托单位: University of East Asia
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660126/
• 关键词: Pseudomonas aerugionsa; alkaline protease; structural gene; secretion gene; site-directed mutagenesis; method of Kunkel; active site; Ca-binding site; 緑膿菌アルカリプロテアーゼ; Zn結合部位; Ca結合部位; 緑膿菌アルカリプロテアーゼ(AP); Western blotting; AP分泌発現系; PCR法

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.

1)铜绿假单胞菌碱性蛋白酶的定点突变为了对铜绿假单胞菌（PA）碱性蛋白酶的活性位点和钙结合位点进行定点突变，从质粒pAPE 1（aprA，PA的结构基因，插入pUC 18）中制备了涉及待突变位点的Kpn 1-Pst 1片段（1.3Kb）。将该片段插入到M13 mp 18载体中，形成单链DNA，按照Kunkel的方法进行定点突变。构建了以下突变体：Zn-配体（H176*L）、催化位点（E177*Q）和Ca-结合位点（D356*A，D365*A）。2）PA分泌型基因的构建我们尝试用PCR方法构建PA分泌型基因（apr D/E/F），但没有成功。因此，以下两种质粒由法国CNRS的Murgier博士提供。pJUEK 72（8.8KB）; 4月D/E/F/A（PA可分泌）pAGS 7（7.0Kb）; 4月D/E/F（仅分泌机制）3）PA定点突变对其在大肠杆菌中分泌的影响用质粒转染大肠杆菌C-600或JM 109（pAGS 7 + pAPE 1或pAGS 7+突变型pAPE 1）观察对分泌的影响。采用LB培养基和1%脱脂乳琼脂平板法，通过晕圈的形成来检测PA的分泌。结果表明，携带pJUEK 72或pAGS 7 + pAPE 1质粒的大肠杆菌均能产生晕轮，而携带突变基因的菌株则不能产生晕轮。对培养滤液进行聚丙烯酰胺凝胶电泳和Western印迹分析，结果表明，突变基因在活性位点上得到了比PA稍大的产物（50 KDa），而在Ca结合位点上得到了小分子的水解产物。对结果进行了讨论。