小分子伴侣协助重组人γ-干扰素体外复性研究  

Minichaperone (GroEL191～345)-mediated In vitro Refolding of Recombinant Human Interferon-γ Inclusion Body

作　　者：关怡新[1] 费峥峥[1] 罗曼[1] 姚善泾[1] 

机构地区：[1]浙江大学化学工程与生物工程学系,杭州310027

出　　处：《生物化学与生物物理进展》2004年第10期907-911,共5页Progress In Biochemistry and Biophysics

基　　金：浙江省自然科学基金 ( 2 0 10 99);教育部留学回国人员科研启动基金资助项目~~

摘　　要：考察了小分子伴侣在游离和固定化两种情况下 ,对重组人γ 干扰素 (rhIFN γ)体外重折叠复性的作用 .实验结果表明 ,小分子伴侣GroEL191～ 345的加入有效地促进了rhIFN γ的复性 ,在初始蛋白质浓度为 10 0mg/L时 ,rhIFN γ复性后蛋白质回收率提高了 2 2倍 ,总活性提高了近 3倍 ;将小分子伴侣固定化在NHS activatedSepharoseFastFlow凝胶后 ,不但能重复利用 ,而且进一步提高了rhIFN γ复性效率 ,在初始蛋白质浓度为4 0 0mg/L时 ,仍使蛋白质回收率达到 4 6 2 9%和比活达到 1 95× 10 7U/mg .The interferons are cytokines with antiviral, antiproliferative and immunomodulatory activities. Among three major distinguished types, interferon-gamma (IFN-γ) is more popular for its specific properties in inhibition of cell growth and modulation of immune functions. Now the recombinant DNA techniques make it possible to express recombinant IFN-γ in E.coli in large amounts, however this may result in the formation of inclusion bodies, and the recovery of biologically active products becomes significant. As the minimal mechanism of GroEL-mediated protein folding, minichaperones, fragments encompassing the apical domain of GroEL, can facilitate the refolding of several proteins in vitro without requiring GroES, ATP, or the cage-like structure of multimeric GroEL. Here minichaperone (GroEL191～345)-mediated in vitro refolding of recombinant human interferon gamma (rhIFN-γ) in free and immobilized GroEL191～345 systems are studied. SDS-PAGE was performed with Laemmli's Tris-glycine buffer system for protein analysis. Protein concentrations were determined by using a modification of Lowry's method with bovine serum albumin as reference and total activities of renatured rhIFN-γ were measured by CPE method. The results showed that the presence of GroEL191～345 in the refolding buffer not only enhanced the specific activity, but also promoted the refolding efficiency. With the initial protein concentration of 100 mg/L, the protein yield and total activity of rhIFN-γ assisted by GroEL191～345 was 2.2 and 3 folds of that under spontaneous condition respectively. Optimal operating parameters in refolding of rhIFN-γ assisted by GroEL191～345 were as follows: refolding temperature 15℃, refolding time 4 h, pH 7.7, initial concentration of IFN-γ 100～200 mg/L and the molar ratio of GroEL 191～345 versus IFN-γ 1∶1～2∶1. Furthermore, the immobilization of GroEL191～345 on NHS-activated sepharose fast flow made it possible to be recycled. The protein yield and the specific activity of rhIFN-γ were 46.29%

关 键 词：小分子伴侣GroEL91~345 重组人Γ-干扰素 复性 包涵体 

分 类 号：Q511[生物学—生物化学]