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Published on: December 30, 2016
[Cloning and expression of protease PT121 from Pseudomonas aeruginosa and application in peptide synthesis]
Objective:
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.
Methods:
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.
Results:
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%.
Conclusions:
Successful hetero-expression of protease PT121 lays the foundation of studying mechanism of catalysis and molecular evolution.
