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Abstract LJAMP2, a member of nonspecific lipid transfer protein, isolated from motherwort seeds, displays antimicrobial activity against fungi. To obtain enough LJAMP2 with activity for performing function analysis in vitro, the LJAMP2 gene was cloned into the pPIC9K vector and transformed into Pichia pastoris using electrotransformation. A transformant with a high copy number of the gene integrated into the chromosome was obtained by YPD plates which contains 1.75 mg/mL G418. The active LJAMP2 was secreted into the culture medium under the induction of 1% methanol and purified more than 90% purity from culture medium by desalting chromatography, CM FF cation exchange chromatography, and reversed-phase HPLC. SDS-PAGE results showed that there was a faint target band in Mut+ recombinant culture supernatant after induction 24 hours culture and the yield of LJAMP2 got to the top after 168 hours culture. The antifungal activity of recombinant LJAMP2 was studied by plate inhibition analysis, which revealed that the recombinant LJAMP2 exhibited activities against Bipolaris maydis and Aspergillus niger. Together, these observations indicate that the recombinant LJAMP2 is an active antifungal protein that can be successfully produced in the yeast, and therefore may be a potential antifungal candidate for practical use.
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Received: 18 September 2009
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