Expression And Bioactivity Detection Of Aiia

The AiiA protein encoded by N-acyl-homoserine lactonase(aiiA) can degrade the AHLs signal molecules, interact the quorum sensing system and attenuate plant disease symptoms caused by baeterial pathogens.An aiiA-expressing vector, pPIC3.5K-aiiA, was constructed and transformed into Pichia pastoris GS115 by electroporation. The recombinant yeast strains were screened with auxotroph medium and phenotypic identification.The transformants with high copy numbers of aiiA gene were selected in medium containing high concentration of G418. The expression of aiiA was induced by addition of methanol into culture at the final concentration of 0.5%. The transcription of aiiA was confirmed by RT-PCR in the recombinant yeast. SDS-PAGE and Western Blot analysis demonstrated that recombinant AiiA protein was successfully expressed after induction. The recombinant AiiA protein showed the activity of degrading the N-acyl-homoserine lactones when using Chromobacterium violaceum CV026 as reporter strain.The recombinant expressing vector pPIC9K-aiiA was construct and transformed into Pichia pastoris strain GS115 by electroporation. The recombinant strain GS115(pPIC9K- aiiA)was chosn by auxotroph medium, phenotypic identification and G418 resistance .After induction by methanol, AiiA were not detected in supernatant or concentrated supernatant. However, the Intracellular material showed activity of degrading the AHLs when using CV026 as reporter strainThe aiiA gene was cloned into express vector pET28a . The recombinant express vector pET28a-aiiA was transformed into Escherichia coli(E.coli) BL21(DE3)to express the recombinant protein under 0.8% IPTG inducing condition. SDS-PAGE and Western Blot analysis showed that recombinant AiiA protein was successfully expressed. Moreover, the recombinant AiiA protein showed the activity of degrading the N-acyl-homoserine lactones when using Chromobacterium violaceum CV026 as reporter strain.