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Expression Of Escherichia Coli Choline Dehydrogenase In Pichia Pastoris

Posted on:2005-05-27Degree:MasterType:Thesis
Country:ChinaCandidate:C M WangFull Text:PDF
GTID:2120360125462460Subject:Cell biology
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Betaine was first noted as an osmoprotectent. Recently, methyl donor role of betaine was further verified by clinical curative effect and biochemical experiment. Osmoprotective activity, methyl donor ability, anticoccidials ability of betaine and its beneficial effect to feed conversion enabled it as an extensively used feed additive. The main purpose of this thesis was to construct an engineered pichia which can express CDH, catalyze betaine synthesis, and can be used as a probiotic strain.Bet A, which encodes CDH, was amplified from Escherichia coli by the method of nested PCR. It was ligated with pUC-T vector and sequenced. Sequence ananlysis with DNA star showed there were 6 nucleotide differenc and 3 amino acid difference compared with the reported sequence, but their hydrophobic property were the same.To express betA in pichia, a shuttle vector pPIC9K, which can express fusion protein both in pichia and E. coli, was constructed. After transformation in E. coli, the constructed vector was extracted and linearized with sad, then transformed into pichia via electroporation at 750V/4.3ms, The transformed clone was plated on MGY medium. The betA gene was integrated into pichia genome verified by PCRamplification. Results of SDS-PAGE showed that CDH was secreted into cultured supernatant under methanol induction. The secreted CDH was precipitated with 50% ammonium sulfate and its enzymatic activity was detected with PMS-Cyt. c as electron donor. Betaine accumulation was detected by HPLC with choline as CDH's substrate.All these results showed the transformed pichia with bet A could secrete the expressed CDH into supernatant. An engineered pichia strain which can express choline dehydrogenase has been successfully constructed.
Keywords/Search Tags:betaine, bet A, CDH, Pichia pastoris
PDF Full Text Request
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