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半胱氨酸蛋白酶IdeS的原核表达、纯化及活性鉴定
许静, 赵自叶, 徐进, 郭怀祖, 郑娟, 段树燕, 彭晓云, 王皓
中国生物工程杂志 ›› 2014, Vol. 34 ›› Issue (10) : 8-14.
PDF(1527 KB)
PDF(1527 KB)
半胱氨酸蛋白酶IdeS的原核表达、纯化及活性鉴定
Prokaryotic Expression,Purification and Activity Identification of Cysteine Hydrolase IdeS
酿脓链球菌来源的免疫球蛋白G降解酶 (immunoglubulin G-degrading enzyme of Streptococcus pyogenes, IdeS) 是一种典型的半胱氨酸水解酶,可以在抗体铰链区的特定位点进行酶切,使IgG水解为完整的 F(ab)2片段和Fc片段,由于其独特的酶活特异性和高活性,IdeS可以作为工具酶应用于IgG的亚单位制备、结构分析及表征分析.利用双标签 (GST-tag及His6-tag) 表达系统在E.coli中高效表达了可溶性重组蛋白酶GST-IdeS-His6,并在IdeS的氨基端与GST之间加上肠激酶酶切位点,以利于标签的去除.再利用亲和层析的纯化方法对目的蛋白进行纯化.使用SDS-PAGE、HPLC-SEC、LC-MS对纯化后的IdeS进行分析和鉴定.结果表明:本系统所表达的蛋白酶IdeS得率高,每1L菌液纯化可获得约25mg纯度为90%以上的蛋白酶IdeS,将此蛋白酶与抗体IgG以1:100 (m/m) 比例混合,于37℃反应30min,即可酶切完全.能够满足蛋白质结构分析中对蛋白酶的高质量要求,并且适用于抗体类药物的表征分析.同时,蛋白酶IdeS也可以应用于生物仿制药、生物改良药和新一代抗体以及Fc-融合蛋白的研究.
ImmunoglobulinG-degrading enzyme of Streptococcus pyogenes (IdeS) is a kind of typical cysteine hydrolase, which cleaves IgG at a specific site in the hinge area and leads to the formation of F(ab)2 and Fc fractions. Because of its unique specificity and hign activity, IdeS could be used in the preparation of subunits, structural analysis and charaterization of IgG. The recombinant GST-IdeS-His6 was efficiently expressed in Escherichia coli by using a two-tag system. And an enteropeptidase cleavage site was inserted at the N-terminal of IdeS for removing the GST-tag easily. The enterokinase site was also added between amino terminal and GST, in order to remove the GST-tag easily. Purified by affinity chromatography, IdeS was identified and analysised by SDS-PAGE, HPLC-SEC and LC-MS.The results suggested that this system was efficient for the production of IdeS. There would be about 25mg protein, with the purity more than 90%, obtained from one liter bacteria liquid. The protease and antibody IgG could be mixed with 1:100 (m/m) ratio, reacted at 37 ℃ for 30 min, then digested completely. The recombinant IdeS could meet the quality requirement of protein structural analysis and could be further utilized effectively in the characterization analysis of antibody drugs. And this enzyme could be also applied in the research of biosimilars, biobetters, and next-generation antibodies and Fc-fusion proteins.
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