Exiguobacterium sp. MY02菌株PyNPase基因的克隆与表达
Clone and expression of a new PyNPase gene of Exiguobacterium sp. MY02
作者：姜立春;韩文君;余蓉;阮期平;
Author：
收稿日期： 年卷（期）页码：2007,(06):-600-604
期刊名称：华西药学杂志
Journal Name：WEST CHINA JOURNAL OF PHARMACEUTICAL SCIENCES
关键字：Exiguobacterium;嘧啶核苷磷酸化酶;高效表达;磷酸化反应
Key words：
基金项目：
中文摘要
目的鉴定、构建并高效表达从土壤中筛选到的一株耐热型、高活性的嘧啶核苷磷酸化酶(PyNPase)。方法以MY02总DNA为模板,通过简并PCR、特异PCR引物、筛选和DNA测序,在5’引物和3’引物中分别引入NcoI和XbaI酶切位点,将pynp基因克隆至分泌表达载体pBAD/gIII A中,转化至E.coliTOP 10F’,以L-阿拉伯糖诱导表达,并对诱导剂浓度、诱导时间等进行优化。结果成功构建了重组表达载体pBAD/gIII A-pynp,通过磷酸化和转化反应,鉴定此酶为PyNPase。结论优化PyNPase的纯化条件与复性条件,可获得大量的PyNPase。
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