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D-氨基酸氧化酶在毕赤酵母中的表达及快速纯化 被引量：2

Expression and quick purification of recombinant D-amino acid oxidase in Pichia pastoris
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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.
作者 冯美卿 史训龙 孙传文 周伟 周珮
机构地区 复旦大学药学院生物合成药物化学教研室
出处 《复旦学报（医学版）》 CAS CSCD 北大核心 2006年第5期622-625,共4页 Fudan University Journal of Medical Sciences
关键词 D-氨基酸氧化酶 毕赤酵母 表达 快速纯化 D-amino acid oxidase Pichia pastoris expression purification
分类号 Q814 [生物学—生物工程]
  • 相关文献

参考文献12

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共引文献4

同被引文献32

  • 1史训龙,冯美卿,贺佳音,袁中一,周珮.基因工程酵母菌来源的D-氨基酸氧化酶分离纯化[J].复旦学报（医学版）,2005,32(1):71-74. 被引量：2
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  • 3孙兵兵,王筱兰,杨志平.D-氨基酸氧化酶的研究进展[J].化学与生物工程,2007,24(2):8-11. 被引量：3
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引证文献2

二级引证文献6

复旦学报（医学版）
2006年 第5期

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