D-氨基酸氧化酶在毕赤酵母中的表达及快速纯化  

Expression and quick purification of recombinant D-amino acid oxidase in Pichia pastoris

作　　者：冯美卿[1] 史训龙[1] 孙传文[1] 周伟[1] 周珮[1] 

机构地区：[1]复旦大学药学院生物合成药物化学教研室,上海200032

出　　处：《复旦学报（医学版）》2006年第5期622-625,共4页Fudan University Journal of Medical Sciences

摘　　要：目的建立简便、快速纯化D-氨基酸氧化酶(DAAO)的新方法,以利用高纯度的DAAO转化头孢菌素C制备戊二酰-7-氨基头孢霉烷酸(GL-7ACA)。方法通过基因工程手段,构建N端和C端各镶嵌6个组氨酸的DAAO重组质粒,电转化Pichia pastoris GS115电感受态细胞,利用PCR方法筛选阳性转化子,再经甲醇诱导筛选出高表达菌。结果高表达重组菌(PHD12)摇瓶发酵单位达到2 000 IU/L,发酵罐内达到22 500 IU/L。并利用Ni螯合型树脂对表达的DAAO经一步分离纯化,以60%的总收率获得纯化产物。纯化产物保持良好的转化CPC-Na活性。结论利用基因工程表达组氨酸标记蛋白,可简化基因工程的下游工序。Purpose To develop a new method to purify D-amino acid oxidase （DAAO） efficiently and fast. Methods Six additional histidine amino acid residues were tagged at N and C-termini of DAAO. His-tagged DAAO gene was cloned into pPIC3.5k of Pichia pastoris. Then it was purified by nickel-chelate chromatography. Results His-tagged protein was successfully expressed in Pichia pastoris GSll5 with 22 500 IU per liter and purified quickly to total yield of 60% by one step. Conclusions His-tagged enzyme still keeps the capability of bioconversion cephalosporin C to 7-glutary cephalosporanic. The purification was more fast and efficiently.

关 键 词：D-氨基酸氧化酶 毕赤酵母 表达 快速纯化 

分 类 号：Q814[生物学—生物工程]