Abstract
BackgroundHuman glucose 6-phosphate dehydrogenase (G6PD), active in both dimer and tetramer forms, is the key entry enzyme in the pentose phosphate pathway (PPP), providing NADPH for biosynthesis and various other purposes, including protection against oxidative stress in erythrocytes. Accordingly haemolytic disease is a major consequence of G6PD deficiency mutations in man, and many severe disease phenotypes are attributed to G6PD folding problems. Therefore, a robust refolding method with high recovery yield and reproducibility is of particular importance to study those clinical mutant enzymes as well as to shed light generally on the refolding process of large multi-domain proteins.ResultsThe effects of different chemical and physical variables on the refolding of human recombinant G6PD have been extensively investigated. L-Arg, NADP+ and DTT are all major positive influences on refolding, and temperature, protein concentration, salt types and other additives also have significant impacts. With the method described here, ~70% enzyme activity could be regained, with good reproducibility, after denaturation with Gdn-HCl, by rapid dilution of the protein, and the refolded enzyme displays kinetic and CD properties indistinguishable from those of the native protein. Refolding under these conditions is relatively slow, taking about 7 days to complete at room temperature even in the presence of cyclophilin A, a peptidylprolyl isomerase reported to increase refolding rates. The refolded protein intermediates shift from dominant monomer to dimer during this process, the gradual emergence of dimer correlating well with the regain of enzyme activity.ConclusionL-Arg is the key player in the refolding of human G6PD, preventing the aggregation of folding intermediate, and NADP+ is essential for the folding intermediate to adopt native structure. The refolding protocol can be applied to produce high recovery yield of folded protein with unaltered properties, paving the way for future studies on clinical G6PD mutants with folding defects and providing a useful model system to study the folding process of oligomeric proteins.
Highlights
Human glucose 6-phosphate dehydrogenase (G6PD), active in both dimer and tetramer forms, is the key entry enzyme in the pentose phosphate pathway (PPP), providing NADPH for biosynthesis and various other purposes, including protection against oxidative stress in erythrocytes
The stability of the protein is thought to be critically dependent on the concentration of NADP+ [14,15], which has been clearly shown to be incorporated into the structure of the folded protein [11,16,17], and impaired binding of this "structural" NADP+ is thought to underlie instability in some clinical mutants
Unfolding of human G6PD Native G6PD (2–4 mg/ml), with free or loosely bound NADP+ previously removed by serial dilution, was denatured by different concentrations of guanidinium hydrochloride (Gdn-HCl) in 50 mM Tris-HCl, pH 7.6, at 30°C and kept reduced by the inclusion of 20 mM DTT
Summary
Human glucose 6-phosphate dehydrogenase (G6PD), active in both dimer and tetramer forms, is the key entry enzyme in the pentose phosphate pathway (PPP), providing NADPH for biosynthesis and various other purposes, including protection against oxidative stress in erythrocytes. Human glucose 6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) catalyses the first and rate-limiting step in the pentose phosphate pathway This pathway provides ribose-5-phosphate (R5P) for the synthesis of nucleotides and generates NADPH, both generally for biosynthesis and for more specialised tasks such as protection against oxidative stress in erythrocytes, where G6PD is particu-. BMC Biotechnology 2009, 9:19 http://www.biomedcentral.com/1472-6750/9/19 larly important as the sole source of NADPH In connection with this latter role, G6PD deficiency is the most common human enzymopathy, affecting about 400 million people [1,2], and well over 160 different mutations have been determined at the DNA level [3,4]. A convenient, robust and reliable refolding method giving high yields for the native enzyme is much needed in order to investigate differences in folding ability between G6PD WT and the mutants
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