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Expression And Enzyme Properties Of Recombinant Thermostable Restriction Endonuclease I-Scel In Pichia Pastoris GS115

Posted on:2012-11-03Degree:MasterType:Thesis
Country:ChinaCandidate:X DengFull Text:PDF
GTID:2230330395987709Subject:Biochemistry and Molecular Biology
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I-Scel is a site-specific homing endonuclease encoded by a mitochondrial intron of Saccharomyces cerevisiae. Intron-encoded endonucleases are proteins that promote the first step in mobility of the intron at the DNA level. They recognize and cleave an intronless allele of their cognate gene to insert a copy of the intron by a double-strand-break repair mechanism that results in the recipient allele also becoming intron-plus. Because of these characterstics, endonuclease I-SceI can become beneficial tools in the high-resolution physical map, analysis of the genomic organization, gene cloning and fixed point induced by recombinant, and in DNA double-strand break repair in a wide variety of biological sysytems.At present, all commercial endonuclease I-SceI was expressed in Escherichia coli. However, subsequent purification steps are complex. In order to improve the expression of I-SceI, Pasteur pichia expression system, which has a lot of advantages, such as high levels of expression, low levels of the self-secreted protein, easy to separation and purification, and high density fermentation, is used to enable I-SceI secretion expression. Firstly, the gene I-SceI(708bp) was synthetized and inserted into Pichia pastoris secretion expression vector pHBM905A. The recombinant plasmid was linearized by endonuclease SalI and then transformed into Pichia pastoris strain GS115by electroporation. The recombinant strains were successfully screened on MD plate and identified by PCR analysis. After incubated in flasks to exponential phase, the expression ofI-SceI was induced by0.5%methanol, followed by collecing of supernatant and SDS-PAGE analysis.The result indicated the protein was expressed. The expression level is about179mg/L. Further experiment indicated I-SceI can cut Fast-spec plasmid at two I-SceI recognization sites specificly, which prives that the expressed protein has biological activity. Altogather, I-SceI in expressed in Pichia pastoris system successfully.Meanwhile, gene Sso7d’s ORF, which from Sulfolobus solfataricus, was fused into3’and5’of endonuclease I-SceI ORF, and Pichia pastoris expression system was employed to express these fusion proteins. Both of them are expressed successfully. Consistanted with recombinant I-SceI, the fusion proteins can cut the I-SceI sites on plasmid Fast-spec, indicating thy also have biological activity. More importantly, the fusion proteins can digest the substrate more efficiently. This may due to the characteristcs of sso7d that change DNA’s space structure, so as to make I-SceI bind easier to the DNA at recognization sites.In further work, we intend to screen Pichia pastoris strains that can express high level of sso7d and I-SceI fusion proteins.
Keywords/Search Tags:Endonucleases, I-SceI, sso7d, thermophilic bacteria, Pichia pastoris, expression, Digestion
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