Li Yan, Ning Yun-shan, Hao Wen-bo, Li Li, Li Ming
Institute of Tropical Medicine, First Military Medical University, Guangzhou 510515, China.
Di Yi Jun Yi Da Xue Xue Bao. 2003 Dec;23(12):1273-6.
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).
The recombinant plasmid GDH/pGEX-4T-1, encoding the full-length GDH gene, was transformed into E.coli BL21 (DE3) to achieve IPTG-induced high 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 and dilution, for refolding of the fusion protein. The refolded GDH/GST was then purified by different chromatographic approaches.
SDS-PAGE analysis showed that the expression GDH/GST fusion protein mounted up to approximately 25% of the total bacterial protein. The dilution was better than the other two methods for the refolding of the fusion protein, with the optimized renaturation condition necessitating the presence of 20 mmol/L Tris-HCl and 1 mmol/L EDTA at pH8.5 with GSSG/GSH ratio of 1 10, which resulted in a recovery rate exceeding 90%. Two-step ion exchange chromotography was optimal for purification of the fusion protein.
The high-purity and biologically active GDH/GST can be acquired by dilution renaturation followed by two-step ion exchange chromatography.
建立恶性疟原虫（FCC1/HN）谷氨酸脱氢酶（GDH）与谷胱甘肽S-转移酶（GST）融合蛋白的复性及纯化方法。
将编码全长GDH基因的重组质粒GDH/pGEX-4T-1转化至大肠杆菌BL21（DE3）中，经异丙基-β-D-硫代半乳糖苷（IPTG）诱导，以包涵体形式高效表达GDH/GST，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行鉴定。用8 mol/L尿素变性后，对包涵体采用3种不同的复性方法进行融合蛋白的重折叠，即Sephacryl S-200层析、透析和稀释法，并采用不同的层析方法对复性后的GDH/GST进行纯化。
SDS-PAGE分析显示，表达的GDH/GST融合蛋白约占细菌总蛋白的25%。对于融合蛋白的重折叠，稀释法优于其他两种方法，优化的复性条件为pH8.5时含20 mmol/L Tris-HCl和1 mmol/L EDTA，氧化型谷胱甘肽/还原型谷胱甘肽（GSSG/GSH）比例为1∶10，回收率超过90%。两步离子交换层析法对融合蛋白的纯化效果最佳。
通过稀释复性后两步离子交换层析可获得高纯度且具有生物学活性的GDH/GST。
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