Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, India 625021.
AMB Express. 2011 Mar 28;1(1):3. doi: 10.1186/2191-0855-1-3.
Metagenomic DNA isolated from goat skin surface was used to construct plasmid DNA library in Escherichia coli DH10B. Recombinant clones were screened for functional protease activity on skim milk agar plates. Upon screening 70,000 clones, a clone carrying recombinant plasmid pSP1 exhibited protease activity. In vitro transposon mutagenesis and sequencing of the insert DNA in this clone revealed an ORF of 1890 bp encoding a protein with 630 amino acids which showed significant sequence homology to the peptidase S8 and S53 subtilisin kexin sedolisin of Shewanella sp. This ORF was cloned in pET30b and expressed in E. coli BL21 (DE3). Although the cloned Alkaline Serine protease (AS-protease) was overexpressed, it was inactive as a result of forming inclusion bodies. After solubilisation, the protease was purified using Ni-NTA chromatography and then refolded properly to retain protease activity. The purified AS-protease with a molecular mass of ~63 kDa required a divalent cation (Co2+ or Mn2+) for its improved activity. The pH and temperature optima for this protease were 10.5 and 42°C respectively.
从山羊皮肤表面分离的宏基因组 DNA 被用于在大肠杆菌 DH10B 中构建质粒 DNA 文库。在脱脂乳琼脂平板上筛选具有功能蛋白酶活性的重组克隆。在筛选了 70000 个克隆后，携带重组质粒 pSP1 的克隆显示出蛋白酶活性。对该克隆中插入 DNA 的体外转座子诱变和测序揭示了一个编码 1890 个碱基对的 ORF，该 ORF 编码一种具有 630 个氨基酸的蛋白质，与 Shewanella sp. 的肽酶 S8 和 S53 枯草杆菌蛋白酶 kexin sedolisin 具有显著的序列同源性。该 ORF 被克隆到 pET30b 中，并在大肠杆菌 BL21 (DE3) 中表达。尽管克隆的碱性丝氨酸蛋白酶 (AS-蛋白酶) 过表达，但由于形成包涵体而没有活性。在溶解后，蛋白酶使用 Ni-NTA 色谱法进行纯化，然后正确复性以保留蛋白酶活性。具有~63 kDa 分子量的纯化 AS-蛋白酶需要二价阳离子 (Co2+ 或 Mn2+) 才能提高其活性。该蛋白酶的最适 pH 和温度分别为 10.5 和 42°C。
