Cloning of Pseudomonas aeruginosa Elastase Gene (PAE) and Its Expression in Pichia pastoris

The Pseudomonas aeruginosa elastase (PAE) gene fragment was amplified by PCR from the genome DNA of a strain of Pseudomonas aeruginosa. The nucleotide sequence of the cloned DNA shared 99% homology with lasB from P. aeruginosa PA01 (GenBank accession No. M19472). The secreted recombinant expression plasmid pPIC3.5K/PAE was constructed and digested with SacⅠand introduced into Pichia pastoris KM71. PCR and phenotype analysis showed that PAE has been integrated into P.pastoris genome. After screening, the 48 high-copy recombinant P.pastoris transformants were obtained. The specific expression protein was secreted into the medium after inducing with methanol. SDS-PAGE results showed that there was one main protein band with molecular weight 34 kD in the fermentation supernation. Activity tests revealed that the specific activity of the recombinant elastase was 1 060 U/mL, which was approximately 26-fold higher than that of elastase obtained from P. aeruginosa.