摘要
目的建立恶性疟原虫谷氨酸脱氢酶(GDH)融合蛋白质的复性和纯化方法。方法将含有恶性疟原虫GDH全长基因的融合蛋白表达质粒GDH/ pGEX-4T-1转化到宿主菌BL21,该GDH/GST融合蛋白在IPTG的诱导下获得高水平的表达,经SDS-PAGE分析表达产物主要为不溶性的包涵体。经超声酶解法、离心、变性剂/去污剂洗涤后获得包涵体。包涵体经8 mol/L尿素变性后分别采用分子筛柱层析、透析和稀释3种方法复性,并通过浊度分析、非还原SDS-PAGE等方法比较、优化复性方法和条件。复性后的GDH/GST融合蛋白经过不同柱层析方法进行纯化。结果经SDS-PAGE分析表明,GDH/GST融合蛋白表达量可占到菌体总蛋白的25%左右;浊度分析表明:稀释复性法优于其它两种方法,同时20 mmol/L Tris-HCl、1 mmol/L EDTA、pH8.5及GSSG/GSH=1∶10为该融合蛋白稀释复性的最佳条件,其复性回收率可达90%以上。复性产物采用二步阴离子交换层析可获得较好的纯化效果。结论通过稀释复性,二步阴离子交换层析可获得高纯度、具有天然空间构象的重组GDH/GST融合蛋白。
Objective To establish the method for renaturation and purification of the fusion protein of Plasmodium falfiparum(FCC1/HN ) glutamate dehydrogenase (GDH) with glutathione S-transferase (GST). Methods The recombinant plasmidGDH/pGEX-4T-1, encoding the full-length GDH gene, was transformed into E.coli BL21 (DE3) to achieve IPTG-inducedhigh expression of GDH/GST in the form of inclusion bodies identified by SDS-PAGE.After denaturation with 8 mol/L urea,the inclusion bodies were subjected to 3 different renaturation methods, namely Sephacryl S-200 chromatography, dialysis anddilution, for refolding of the fusion protein. The refolded GDH/GST was then purified by different chromatographic approaches.Results SDS-PAGE analysis showed that the expression GDH/GST fusion protein mounted up to approximately 25% of thetotal bacterial protein. The dilution was better than the other two methods for the refolding of the fusion protein, with theoptimizedrenaturationconditionnecessitatingthepresenceof 20mmol/LTris-HCland 1mmol/LEDTAatpH8.5withGSSG/GSH ratio of 1∶10, which resulted in a recovery rate exceeding 90%. Two-step ion exchange chromotography wasoptimal for purification of the fusion protein. Conclusion The high-purity and biologically active GDH/GST can be acquiredby dilution renaturation followed by two-step ion exchange chromatography.
出处
《第一军医大学学报》
CSCD
北大核心
2003年第12期1273-1276,共4页
Journal of First Military Medical University
基金
广州市科技计划项目(2002E3-E4012)~~
关键词
恶性疟原虫
谷氨酸脱氢酶
融合蛋白
复性
纯化
包涵体
Plasmodium falciparum
glutamate dehydrogenase
fusion protein
inclusion body
renaturation
purification
