Cloning of alkaline protease gene from alkalophilic bacillus strain

Abstract

To clone the alkaline protease gene from alkalophilic Bacillus in E. coli JM107, we prepared a subgenomic library of chromosomal DNA. The chromosomal DNA was digested with EcoRI and fractionated by electrophoresis in a 0.8\% agarose gel, the fragments were joined with the linearized pUC18 vector. The recombinant plasmid was used to transform E. coli JM 107. About 6000 transformants were toothpicked on LBMG plate, and one colony formed clear zone. The recombinant plasmid was named pAL 37 and the insert size was 1.85Kb. And then optimum pH of the alkaline protease was 12. From restriction enzyme mapping, it was found that the insert has AvaII(2), Bgl II(1), EcoRI(1) KpnI(1), NdeI(1) sites.