|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1996 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1995 : ¥1,300,000 (Direct Cost : ¥1,300,000)
1) Site-directed mutagenesis of Pseudomonas aeruginosa alkaline protease
To 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 PA
We 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 coli
The 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.