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

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.

Objectives

To establish a simple, efficient, and optimized method to produce a G6PD_Viangchan variant and characterize the phenotypes of recombinant human wild-type G6PD and G6PD_Viangchan.

Methods

G6PD was amplified by polymerase chain reaction (PCR) from a human cDNA plasmid, and the gene for G6PD_Viangchan 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.

Results

Both 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 G6PD_Viangchan (81.85 U/mg), which is consistent with earlier reports. Analysis in silico showed that the coding change in G6PD_Viangchan has a substantial effect on protein folding structure.

Conclusions

We successfully cloned, expressed, and purified both wild-type G6PD and G6PD_Viangchan proteins. Such a protocol may be useful for creating a model system to study G6PD deficiency disease.

Language:: English

Publication timeframe:: 6 times per year
Journal Subjects:: Medicine, Assistive Professions, Nursing, Basic Medical Science, Basic Medical Science, other, Clinical Medicine, Clinical Medicine, other

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

Lelamekala Vengidasan

Muhammad Amir Yunus

Narazah Mohd Yusoff

Badrul Hisham Yahaya

Ida Shazrina Ismail

Article Category: Technical report

Published Online: Sep 20, 2020

Page range: 159 - 167

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

Keywordsphenotype, 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.

Background

Objectives

Methods

Results

Conclusions

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

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