<abstract xmlns="http://www.w3.org/1999/xhtml"><sec><h3>Background</h3><p>Glucose-6-phosphate dehydrogenase (G6PD) is essential to produce reduced nicotinamide adenine dinucleotide phosphate, which is required to protect cells against oxidative stress. G6PD deficiency is a genetic variation that may lead to hemolysis with potential consequences, such as kidney failure, and patients often experience low quality of life.</p></sec><sec><h3>Objectives</h3><p>To establish a simple, efficient, and optimized method to produce a G6PD<sub>Viangchan</sub> variant and characterize the phenotypes of recombinant human wild-type G6PD and G6PD<sub>Viangchan</sub>.</p></sec><sec><h3>Methods</h3><p><italic>G6PD</italic> was amplified by polymerase chain reaction (PCR) from a human cDNA plasmid, and the gene for G6PD<sub>Viangchan</sub> was amplified by initiating a mutation at location 871 (G>A) through site-directed mutagenesis. Protein expression and western blotting were conducted after successful cloning. The enzymatic activity of both proteins was assessed spectrophotometrically after purification.</p></sec><sec><h3>Results</h3><p>Both amplicons were successfully cloned into a pET26b(+) expression vector and transformed into <italic>Escherichia coli</italic> BL21 (DE3) cells for overexpression as C-terminally histidine-tagged recombinant proteins. Western blotting confirmed that both proteins were successfully produced at similar levels. The enzymes were purified by immobilized metal (Co) affinity chromatography. Postpurification assay of enzyme activity revealed about 2-fold differences in the levels of specific activity between the wild-type G6PD (155.88 U/mg) and G6PD<sub>Viangchan</sub> (81.85 U/mg), which is consistent with earlier reports. Analysis in silico showed that the coding change in G6PD<sub>Viangchan</sub> has a substantial effect on protein folding structure.</p></sec><sec><h3>Conclusions</h3><p>We successfully cloned, expressed, and purified both wild-type G6PD and G6PD<sub>Viangchan</sub> proteins. Such a protocol may be useful for creating a model system to study G6PD deficiency disease.</p></sec></abstract>

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) is essential to produce reduced nicotinamide adenine dinucleotide phosphate, which is required to protect cells against oxidative stress. G6PD deficiency is a genetic variation that may lead to hemolysis with potential consequences, such as kidney failure, and patients often experience low quality of life.ObjectivesTo establish a simple, efficient, and optimized method to produce a G6PDViangchan variant and characterize the phenotypes of recombinant human wild-type G6PD and G6PDViangchan.MethodsG6PD was amplified by polymerase chain reaction (PCR) from a human cDNA plasmid, and the gene for G6PDViangchan was amplified by initiating a mutation at location 871 (G>A) through site-directed mutagenesis. Protein expression and western blotting were conducted after successful cloning. The enzymatic activity of both proteins was assessed spectrophotometrically after purification.ResultsBoth amplicons were successfully cloned into a pET26b(+) expression vector and transformed into Escherichia coli BL21 (DE3) cells for overexpression as C-terminally histidine-tagged recombinant proteins. Western blotting confirmed that both proteins were successfully produced at similar levels. The enzymes were purified by immobilized metal (Co) affinity chromatography. Postpurification assay of enzyme activity revealed about 2-fold differences in the levels of specific activity between the wild-type G6PD (155.88 U/mg) and G6PDViangchan (81.85 U/mg), which is consistent with earlier reports. Analysis in silico showed that the coding change in G6PDViangchan has a substantial effect on protein folding structure.ConclusionsWe successfully cloned, expressed, and purified both wild-type G6PD and G6PDViangchan proteins. Such a protocol may be useful for creating a model system to study G6PD deficiency disease.

Production and differential activity of recombinant human wild-type G6PD and G6PD<sub>Viangchan</sub>

Asian Biomedicine

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Production and differential activity of recombinant human wild-type G6PD and G6PD

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) is essential to produce reduced nicotinamide adenine dinucleotide phosphate, which is required to protect...

Production and differential activity of recombinant human wild-type G6PD and G6PD_Viangchan

Article Category: Technical report

Publié en ligne: 20 sept. 2020

Pages: 159 - 167

DOI: https://doi.org/10.1515/abm-2020-0023

Mots clés
phenotype, glucose-6-phosphate dehydrogenase Viangchan, human, cloning, protein expression, glucosephosphate dehydrogenase deficiency

© 2020 Lelamekala Vengidasan et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Production and differential activity of recombinant human wild-type G6PD and G6PDViangchan

Article Category: Technical report

Publié en ligne: 20 sept. 2020

Pages: 159 - 167

DOI: https://doi.org/10.1515/abm-2020-0023

Mots clésphenotype, glucose-6-phosphate dehydrogenase Viangchan, human, cloning, protein expression, glucosephosphate dehydrogenase deficiency

© 2020 Lelamekala Vengidasan et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Production and differential activity of recombinant human wild-type G6PD and G6PD_Viangchan

Mots clés
phenotype, glucose-6-phosphate dehydrogenase Viangchan, human, cloning, protein expression, glucosephosphate dehydrogenase deficiency