Construction of an aiiA-expressing Vector and Its Expression in Pichia pastoris

Abstract:

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.

Key words:aiiA gene, Pichia pastoris, Gene expression