title: [Cloning and expression of protease PT121 from Pseudomonas aeruginosa and application in peptide synthesis].
abstract: 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.
authors.forename: Fucheng
authors.initials: F
authors.lastname: Zhu
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhuang
authors.forename: Bingfang
authors.initials: B
authors.lastname: He
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; D010447:Peptide Hydrolases