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

Lelamekala Vengidasan
Lelamekala Vengidasan
, 
Muhammad Amir Yunus
Muhammad Amir Yunus
, 
Narazah Mohd Yusoff
Narazah Mohd Yusoff
, 
Badrul Hisham Yahaya
Badrul Hisham Yahaya
 e 
Ida Shazrina Ismail
Ida Shazrina Ismail
   | 20 set 2020
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Article Category: Technical report
Pubblicato online: 20 set 2020
Pagine: 159 - 167
DOI: https://doi.org/10.1515/abm-2020-0023
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© 2020 Lelamekala Vengidasan et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 1

Agarose gel electrophoresis showing purified 5.9 kb vector pET26b (lane V), the PCR-amplified glucose-6-phosphate dehydrogenase gene (G6PD) 1.5 kb insert (lane I), and the plasmid DNA isolated from positive transformation clones (lanes C2, C3, C4, C9, and C10), which was digested using NdeI and XhoI. Lane L is a 1 Kb Plus DNA Ladder (Thermo Fisher Scientific; catalog No. 10787018). DNA (100 ng) was quantified spectrophotometrically using Nanodrop 2000C Spectrophotometer (Thermo Scientific) and mixed with 1× DNA Gel Loading Dye (Thermo Fisher Scientific) before loading onto a 1% agarose gel (7 cm × 10 cm) and separated by electrophoresis at 120 V for 45 min in 1 × TAE buffer (40 mM Tris-acetate and 1 mM EDTA at pH 8.3) in a Mini-Sub cell GT horizontal gel electrophoresis system (Bio-Rad). The separated DNA was stained with 0.5 μg/mL of ethidium bromide, and visualized under ultraviolet light at 300–360 nm. The isolated plasmid DNA produced 2 products when digested indicating successful cloning of G6PD into the vector. The purified products were used in the subsequent ligation reaction.
Agarose gel electrophoresis showing purified 5.9 kb vector pET26b (lane V), the PCR-amplified glucose-6-phosphate dehydrogenase gene (G6PD) 1.5 kb insert (lane I), and the plasmid DNA isolated from positive transformation clones (lanes C2, C3, C4, C9, and C10), which was digested using NdeI and XhoI. Lane L is a 1 Kb Plus DNA Ladder (Thermo Fisher Scientific; catalog No. 10787018). DNA (100 ng) was quantified spectrophotometrically using Nanodrop 2000C Spectrophotometer (Thermo Scientific) and mixed with 1× DNA Gel Loading Dye (Thermo Fisher Scientific) before loading onto a 1% agarose gel (7 cm × 10 cm) and separated by electrophoresis at 120 V for 45 min in 1 × TAE buffer (40 mM Tris-acetate and 1 mM EDTA at pH 8.3) in a Mini-Sub cell GT horizontal gel electrophoresis system (Bio-Rad). The separated DNA was stained with 0.5 μg/mL of ethidium bromide, and visualized under ultraviolet light at 300–360 nm. The isolated plasmid DNA produced 2 products when digested indicating successful cloning of G6PD into the vector. The purified products were used in the subsequent ligation reaction.

Figure 2

Sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) of unpurified and purified glucose-6-phosphate dehydrogenase (G6PD). After electrophoresis, the proteins in the 12.5% polyacrylamide resolving gel were stained with Coomassie Blue R. The purified G6PD has a molecular mass of 59 kDa. Lane 1: Benchmark Pre-Stained Protein Ladder (Invitrogen, catalog No. 10748010). Lane 2: unpurified wild-type protein. Lane 3: purified wild-type protein. Lane 4: unpurified Viangchan variant protein. Lane 5: purified Viangchan variant protein).
Sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) of unpurified and purified glucose-6-phosphate dehydrogenase (G6PD). After electrophoresis, the proteins in the 12.5% polyacrylamide resolving gel were stained with Coomassie Blue R. The purified G6PD has a molecular mass of 59 kDa. Lane 1: Benchmark Pre-Stained Protein Ladder (Invitrogen, catalog No. 10748010). Lane 2: unpurified wild-type protein. Lane 3: purified wild-type protein. Lane 4: unpurified Viangchan variant protein. Lane 5: purified Viangchan variant protein).

Figure 3

Western blot analysis of lysates containing unpurified and purified glucose-6-phosphate dehydrogenase (G6PD) protein using anti-G6PD (upper panel), anti-6 × HisTag (lower panel) and anti-β-actin antibodies (both panels). A distinct single band is visible indicating immunoreactivity of target proteins to anti-G6PD, and anti-6 × His-Tag antibodies. No band is visible when purified G6PD protein was incubated with anti-β-actin antibody indicating the G6PD protein was purified successfully. Lane M: Benchmark Pre-Stained Protein Ladder (Invitrogen, catalog No. 10748010; markers shown are 82, 64, 49, 37 kDa). Lane 1: unpurified wild-type enzyme. Lane 2: purified wild-type enzyme. Lane 3: unpurified Viangchan variant. Lane 4: purified Viangchan variant).
Western blot analysis of lysates containing unpurified and purified glucose-6-phosphate dehydrogenase (G6PD) protein using anti-G6PD (upper panel), anti-6 × HisTag (lower panel) and anti-β-actin antibodies (both panels). A distinct single band is visible indicating immunoreactivity of target proteins to anti-G6PD, and anti-6 × His-Tag antibodies. No band is visible when purified G6PD protein was incubated with anti-β-actin antibody indicating the G6PD protein was purified successfully. Lane M: Benchmark Pre-Stained Protein Ladder (Invitrogen, catalog No. 10748010; markers shown are 82, 64, 49, 37 kDa). Lane 1: unpurified wild-type enzyme. Lane 2: purified wild-type enzyme. Lane 3: unpurified Viangchan variant. Lane 4: purified Viangchan variant).

Figure 4

Three-dimensional structure of glucose-6-phosphate dehydrogenase (G6PD)Viangchan obtained through mutation in silico. The wild-type G6PD structure (PDBID: 2HB9) was retrieved from the RCSB Protein Data Bank and subjected to mutation in silico using the UCSF Chimera Rotamer tool [25]. Valine 291 was changed to methionine (in red) (G6PDViangchan). The side chain rotamer of the mutated residue was selected based on the Dynameomics rotamers library [26]. The energy minimization was performed on the G6PDViangchan variant (V291M) using the Gromacs simulation package (version 4.6.7) [27]. The molecular docking was performed using AutoDock (version 4.2) [28]. The yellow region represents the structural nicotinamide adenine dinucleotide phosphate (NADP+) binding site and green region represents substrate binding site.
Three-dimensional structure of glucose-6-phosphate dehydrogenase (G6PD)Viangchan obtained through mutation in silico. The wild-type G6PD structure (PDBID: 2HB9) was retrieved from the RCSB Protein Data Bank and subjected to mutation in silico using the UCSF Chimera Rotamer tool [25]. Valine 291 was changed to methionine (in red) (G6PDViangchan). The side chain rotamer of the mutated residue was selected based on the Dynameomics rotamers library [26]. The energy minimization was performed on the G6PDViangchan variant (V291M) using the Gromacs simulation package (version 4.6.7) [27]. The molecular docking was performed using AutoDock (version 4.2) [28]. The yellow region represents the structural nicotinamide adenine dinucleotide phosphate (NADP+) binding site and green region represents substrate binding site.

Specific enzyme activity (U/mg) of heterologous glucose-6-phosphate dehydrogenase

VariantRoos et al. [18]Wang et al. [20]Huang et al. [19]Wang and Engel [21]Gómez-Manzo et al. [33]Gómez-Manzo et al. [17]Gómez-Manzo et al. [23]Boonyuen et al. [22]
Wild-type G6PD210180182210224224230228
Amsterdam–––95––––
Volendam36–––––––
Wisconsin––––178–––
Nashville–130–––103––
Yucatan–––––132––
Valladolid–––––92––
Mexico City–––––175––
Mahidol––177––––141
Fukaya–175––––––
Champinas–160––––––
Plymouth––187–––––
Viangchan––––––228107
Zacatecas––––––58–
Vanua-Lava––––––182–
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