[Refolding and purification of Plasmodium falciparum glutamate dehydrogenase fusion protein].
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 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.


RESULTS
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.


CONCLUSIONS
The high-purity and biologically active GDH/GST can be acquired by dilution renaturation followed by two-step ion exchange chromatography.
Li
Yan
Y
Institute of Tropical Medicine, First Military Medical University, Guangzhou 510515, China.
Ning
Yun-shan
YS
Hao
Wen-bo
WB
Li
Li
L
Li
Ming
M
D000818:Animals; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000737:chemistry*; D005982:Glutathione Transferase / Q000737:chemistry; D010963:Plasmodium falciparum / Q000201:enzymology*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D005969:Glutamate Dehydrogenase; D005982:Glutathione Transferase