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| Purification, molecular gene cloning of an Antimicrobial Protein from Seeds of Ginkgo biloba L and Its Prokaryotic Expression |
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Abstract An antimicrobial protein, with an apparent molecular mass of 12 kD, was isolated from the seeds of Ginkgo biloba. The isolation procedure entailed extraction, ammonium sulfate precipitation, cation exchange chromatography on CM Sephorose F.F, gel filtration chromatography on Superdex 75. MALDI-TOF-MS PMF identification showed higher homology with a novel antifungal protein Ginkbilobin-2. We have designed degenerate primers to clone a fragment of the antimicrobial protein gene, according to the ESI-MS/MS sequence results of internal peptide. The amplified fragment also showed 99% similarity with the Ginkbilobin-2 gene. Further, the homological primers were designed for cloning the full length cDNA of the antimicrobial protein by RT-PCR. The sequence has been deposited in the GenBank (Accession No.FJ865399). In addition, we have constructed the prokaryotic expression vector pGEX-GK2-1 which was used to express the antimicrobial protein in vitro. Results of SDS-PAGE and Western blot showed that the recombinant protein with the calculated molecular mass of 38kD was highly expressed in Escherichia coli BL21(DE3). Our studies lay a foundation for the further research of the antimicrobial mechanism and obtaining the transgenic disease-resistant germplasm resources.
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Received: 01 April 2009
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