Zhu Fucheng, Zhuang Yu, He Bingfang
Wei Sheng Wu Xue Bao. 2015 Jan 4;55(1):67-72.
We studied the cloning and expression of lasB encoding solvent-resistant protease from Pseudomonas aeruginosa PT121. The recombinant protease was then characterized and applied in peptide synthesis.
The PCR primers were designed to acquire the open read frame (ORF) of lasB according to similar protease gene (pseudolysin) reported in the literature. Inducible expression plasmid pET22b-lasB' was constructed and expressed in E. coli BL21 (DE3). The recombinant protease was then characterized and applied in peptide synthesis.
The protease PT121 was defined as metalloproteinase M4 family according to sequence blast. Gene sequence analysis shows that lasB encodes signal peptide, pro-peptide and mature peptide. Mature protein contains 301 residues with molecular weight of 33 kDa. One-step preparation of the recombinant proteases PT121 was optimized by breaking cell wall. The specific activity of protease PT121 reached up to 7700U/mg, and it was stable similar with wild type PT121 from P. aeruginosa PT121 in temperature, pH and organic solvent. The synthesis rate of various dipeptides in 50% DMSO was effective, especially productivity of aspartame precursor reached up to 91%.
Successful hetero-expression of protease PT121 lays the foundation of studying mechanism of catalysis and molecular evolution.
我们研究了铜绿假单胞菌PT121中编码耐溶剂蛋白酶的lasB的克隆与表达。随后对重组蛋白酶进行了表征并将其应用于肽合成。
根据文献报道的相似蛋白酶基因（假溶素）设计PCR引物，以获取lasB的开放阅读框（ORF）。构建可诱导表达质粒pET22b-lasB'并在大肠杆菌BL21（DE3）中表达。随后对重组蛋白酶进行了表征并将其应用于肽合成。
根据序列比对，蛋白酶PT121被定义为金属蛋白酶M4家族。基因序列分析表明，lasB编码信号肽、前肽和成熟肽。成熟蛋白含有301个残基，分子量为33 kDa。通过破壁优化了重组蛋白酶PT121的一步制备方法。蛋白酶PT121的比活性高达7700U/mg，在温度、pH和有机溶剂方面与来自铜绿假单胞菌PT121的野生型PT121相似，具有稳定性。在50%二甲基亚砜中各种二肽的合成率有效，尤其是阿斯巴甜前体的产率高达91%。
蛋白酶PT121的成功异源表达为研究催化机制和分子进化奠定了基础。
