纯化大肠杆菌表达胆固醇氧化酶的两种亲和分离介质的研究

The Research of Two Affinity Mediums for the Purification of a Cholesterol Oxidase(COD) Expression in Escherichia coli

建立了从重组菌中高效亲和制备胆固醇氧化酶的方法,将一种源自Brevibacterium sp.(DQ345780)的胆固醇氧化酶基因转入Escherichia co●BL21(DE3)表达,,选择核黄素-5'磷酸(FMN)及7-氯-异咯嗪作为亲和配体,构建胆固醇氧化酶亲和制备介质。通过两种介质的一步亲和吸附,获得纯度较高的胆固醇氧化酶样本,蛋白回收率分别为9.4%与9.9%(质量百分比),活性回收率分别为85.2%与93.4%(活性百分比)。使用SDS-PAGE分析,纯化得到蛋白分子量为约50 000,纯度分别为98.0%与97.5%(纯度百分比)。通过静态吸附分析,胆固醇氧化酶相对两种亲和介质的最大理论吸附值分别为71.0与78.5 mg/g介质;解离常数分别为12.8和7.3μg/g介质。

In this study,affinity protocols were developed for the preparation of cholesterol oxidase (COD) from recombinant bacteria,a COD gene from Brevibacterium sp.(DQ345780) was expressed in Escherichia coli BL21(DE3),Riboflavin 5'-phosphate and 7-chroroalloxazine were chosen as the affinity ligands,and they were coupled with Sepharose CL 4B through spacers. After one step of affinity binding with the two mediums,the enzyme could be extracted with high purity.The yields of the enzyme purified with the two mediums were 9.4%and 9.9%,respectively,and the recoveries of typical cholesterol oxidase activity were 85.2%and 93.4%.The purified cholesterol oxidases were 98.0%and 97.5%pure with SDS-PAGE analysis. On SDS-PAGE gel,the enzyme was a single polypeptide with the mass of～50 kDa.The theoretical maximum absorption Qmax were 71.0 and 78.5 mg/g medium;the desorption constant K_d of the two mediums on the mediums were 12.8 and 7.3 g/g medium.