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Abstract In this study, the genome DNA of a strain of Pseudomonas aeruginosa was used as the template, via PCR, a 1.67kb product was obtained. We discovered the nucleotide sequence of the cloned DNA shared 99% homology with lasB from P. aeruginosa PA01 (GenBank accession no. M19472). Then the correct plasmid was digested with BamHI and EcoRI and ligated into pPIC3.5K with the same enzymic sites, generating pPIC3.5K/PAE. The insertion was identified by restriction analysis and sequencing.pPIC3.5K/PAE was linearized with Sac I, and electroporated into P. pastoris KM71. Nearly 400 transformants were selected on the MD agar plates，and high-copy transformant weas screened by YPD plates containing G418.Transformant stains were further confirmed by genome PCR and casein-MM plate. After expression of recombinant elastase in shaking flask by methanol induction, we found the size of recombinant elastase was similar to that of the native elastase (approximately 34 kDa) by SDS-PAGE analysis. The highest expression level of the recombinant elastase obtained is approximately 400 mg/L and the specific activity of the recombinant elastase was 1060 U/ml, which was approximately 26-fold higher than that of elastase obtained from P. aeruginosa. The Pseudomonas aeruginosa elastase gene was successfully cloned in this study, which has laid a foundation for high level expression of elastase.
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Received: 13 October 2008
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Corresponding Authors:
韦露450103198306212562李丽婷120103198310162928
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