Cloning, Sequencing and Expression of the Alkaline Protease Gene from Bacillus licheniformis 2709
地衣芽孢杆菌2709碱性蛋白酶基因的克隆、序列测定及其表达

Using the PCR amplified partial coding sequence of the alkaline protease (subtilisin) from Bacillus licheniformis 2709 as the probe of in situ hybridization,several positive colonies were identified from the 2709 genomic library. Two of them , pSC1 and pSC7, were shown having the intact 2709 gene by physical mapping and partial sequencing. The complete sequence of the insert of pSC7 plasmid has 2271 base pairs including 299bp 5' flanking sequence, 832bp 3'flanking sequence and 1140bp structural sequence. The 2709 subtilisin sequence obtained here is highly homologous to the subtilisin from B. licheniformis NCIB 6816. The cloned gene was introduced into the protease-deficient Bacillus subtilis DB104 and protease activity assay demonstrated that a successful expression of 2709 subtilisin in DB104 was achieved.