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Extraction Of CEL I Endonuclease From Apium Graveolens And Expression Of Recombinant CEL I Protein In Pichia Pastoris

Posted on:2011-03-03Degree:MasterType:Thesis
Country:ChinaCandidate:G K K ShangFull Text:PDF
GTID:2120360305974377Subject:Biochemistry and Molecular Biology
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TILLING (Targeting Induced Local Lesions IN Genomes ) is a high-throughput ,rapid and low-cost powerful tool and is widely used for reverse genetics research. The core technology in TILLING is screening mutant library by using single-strand endonuclease. As the the first discovered eukaryotic nuclease, CEL I endonuclease, isolated from celery Apium graveolens, is a mismatch specific endonuclease capable of detecting induced or natural variation in genes of interest. CEL I endonuclease cleaves DNA with high specificity at sites of mismatches. The widely use of TILLING technology requires large quantity of high quality CEL I endonuclease. In this study, the methylotrophic yeast Pichia pastoris was employed to express recombinant CEL I protein. P. pastoris has been used for successful expression of over 300 proteins science 1988 and the full length CEL I gene of A. graveolens is available (GeneBank accession number AF237958). Crude CEL1 protein extract from A. graveolens was used to optimize screening of EMS mutants of Phytophthora sojae.The full length coding region of 888bp of CEL I endonuclease gene was successfully amplified from A. graveolens by RT-PCR, and cloned into vectors pPIC9K and pPICZ╬▒B with a fusion of 6xHis-tag . The recombinant vectors were introduced into P. pastoris strains KM71H and GS115. The expression of recombinant protein in P. pastoris was induced by methanol. SDS-PAGE assay showed the presence of an expected 45kDa protein from the supernatant, and the result was further confirmed by Western blot analyses, showing that the CEL I protein was successfully expressed in P. pastoris and secreted into the culture medium.The active crude CEL1 extract was successfully obtained from A. graveolens and was used to screen for mutants of P. sojae PsPMA1 and an optimized mutant screening procedure was established for tilling target genes in the P. sojae mutants.
Keywords/Search Tags:CEL I Endonuclease, Pichia pastoris, Recombinant protein expression, Western blot, mutant screening
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