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

doi:10.1515/abm-2020-0023

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

Asian Biomed (Res Rev News). 2020 Sep 20;14(4):159-167. doi: 10.1515/abm-2020-0023. eCollection 2020 Aug.

Authors

Lelamekala Vengidasan¹, Muhammad Amir Yunus², Narazah Mohd Yusoff¹, Badrul Hisham Yahaya¹, Ida Shazrina Ismail¹

Affiliations

¹ Regenerative Medicine Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Malaysia, Bertam13200, Penang, Malaysia.
² Infectomics Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam13200, Penang, Malaysia.

Abstract

Background: 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.

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