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

G6PD was amplified by polymerase chain reaction (PCR) using pOTB7 cloning vector as a template, which was purchased from the Mammalian Gene Collection (MGC) of cDNA clones (Dharmacon). Restriction sites NdeI and XhoI were inserted (as underlined) in the PCR primers sequence 5′-TTTCATATGGCAGAGCAGGTGG-3′ (forward) and 3′-CCCCCACAAGCTCGAGAAA-5′ (reverse). PCR was performed under the following conditions: denaturation at 95°C for 5 min, amplification at (95°C for 15 s, 60°C for 30 s, and 68°C for 90 s) for 25 cycles, and 10 min of extension at 72°C. The PCR Master Mix comprising 1 × Pfx amplification buffer, 0.3 mM dNTP mixture, 1 mM MgSO₄, 0.3 μM of primer mix, and 1 U of Platinum Pfx DNA polymerase (Invitrogen) was used to produce approximately 1.5 kb amplicon. PCR was performed by using an Applied Biosystems Veriti 96-Well Thermal Cycler (ThermoScientific).

The PCR product and pET26b(+) (Novagen) were digested using NdeI (New England Biolabs) and XhoI (New England Biolabs) restriction enzymes for 2 h at 37°C in CutSmart Buffer (New England Biolabs). The digested products were purified by electrophoresis in 1% agarose gel (Figure 1) and ligated at 22°C for 15 min. The ligation mix was transformed into DH5α Competent Cells (New England Biolabs) and plated on kanamycin-resistance selective agar plates followed by incubation overnight at 37°C.

Figure 1

The mutation was initiated in wild-type G6PD/pET26 (+) plasmid using a QuickChange Site-Directed Mutagenesis Kit (Agilent). Primers were designed using software: (http://www.genomics.agilent.com/primerDesignProgram.jsp) that is, 5′-CTGAGATGCATTTCAACATCTTGACCTTCTCATCACG-3′ (forward) and 5′-CGTGATGAGAAGGTCAAGATGTTGAAATGCATCTCAG-3′ (reverse). The PCR mixture comprised 50 ng DNA template, 125 ng forward primer, 125 ng reverse primer, 0.3 mM dNTP mixture, 1 × reaction buffer, 2.5 U of PfuUltra HF DNA Polymerase (Agilent) and water, in a final volume reaction of 25 μL. The PCR cycling parameters used were 1 cycle of denaturation at 95°C for 30 s followed by 12 cycles of amplification at 95°C for 30 s, 55°C for 1 min, and 68°C for 4 min. The PCR product (of approximately 1.5 kb) was digested for 1 h using 1 U of DpnI (10 U/μL) (ThermoScientific). The digested product was then transformed into XL1-Blue Competent Cells (Agilent) and then plated on chloramphenicol-resistance (Sigma-Aldrich) selective agar plates for overnight incubation at 37°C. Positive plasmid was sequenced to confirm the mutated site.

Genes for wild-type G6PD and G6PD_Viangchan were transformed into Escherichia coli BL21 (DE3) competent cells for protein expression. At log phase, OD_600nm 0.8, the culture was induced with 0.1 mM and 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and grown at 16°C, 25°C, and 37°C for 3, 5, 12, and 16 h. Cell lysate was centrifuged at 4,000 rpm for 10 min using an Eppendorf Benchtop 5424 Microcentrifuge with an FA-45-11 rotor. The pellets were resuspended with lysis buffer (0.1 M Tris-HCl, pH 7.6, 3 mM MgCl₂, 0.5 mM EDTA free protease inhibitor cocktail (Merck), and 0.1% β-mercaptoethanol). The cells were sonicated on ice with 10 short pulses (10 s) followed by pauses (30 s). The suspension was centrifuged at 15,000 rpm using an Eppendorf Benchtop 5424 Microcentrifuge with an FA-45-11 rotor for 15 min at 4°C. Supernatants were collected as soluble protein. Protein concentration was determined using a Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific) with bovine serum albumin as the protein standard, before sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. All G6PD proteins expressed were C-terminally tagged with histidine (His-Tag).

Proteins (30 μg) were loaded into wells in a 5% polyacryl-amide stacking gel on top of a 12.5% polyacrylamide resolving gel (8.3 cm × 7.3 cm) and separated for 90 min at 180 V in 1 × running buffer (Tris-glycine buffer with 10% SDS) using a Mini-Protean Tetra cell system (Bio-Rad). The proteins in the gel were stained with Coomassie Blue R-250 staining solution for about 30 min and destained using distilled water.

For western blotting analysis, the protein in an unstained gel was transferred to a polyvinylidene difluoride (PVDF) membrane in a semidry manner using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (catalog No. 170-3940; Bio-Rad) in transfer buffer (containing 25 mM Tris, 192 mM glycine, 20% (v/v) methanol (pH 8.3)) at 18 V for 1 h. Non-specific binding to the membrane was then blocked with 5% skimmed milk in phosphate buffered saline (PBS) for 1 h at room temperature. Subsequently, the membrane was incubated with polyclonal rabbit anti-G6PD (1:1,000 dilution; Cell Signaling Technologies, catalog No. 8866S), polyclonal rabbit anti-6′HisTag (1:1,000 dilution; Abcam, catalog No. ab14923) and polyclonal rabbit anti-β-actin (1:1,000 dilution; Abcam, catalog No. ab8227) primary antibodies, overnight at 4 °C. The primary antibodies were diluted in PBS with 5% skimmed milk and 0.1% Tween-20. After washing several times in PBS, the membrane was incubated with goat anti-rabbit IgG (H and L) secondary antibody conjugated to horseradish peroxidase (HRP) (1:10,000 dilution; Abcam catalog No. ab6721). β-Actin protein served as a loading control to show that equal amounts of protein was loaded into the wells. Benchmark Pre-Stained Protein Ladder (Invitrogen, catalog No. 10748010) was used to provide size markers. Finally, enhanced chemiluminescence (ECL) Western Blotting Detection Reagents (GE Healthcare) were used to detect the protein bands and the image was viewed and documented using a Versadoc Imaging system (Bio-Rad).

G6PD was purified using a HisTALON Gravity Column Purification kit (TaKaRa Clontech). As a starting material, we used 10 g of cell pellet. The pellet was suspended in 5 mL of lysis buffer and incubated on ice for 15 min. The suspension was sonicated on ice with 10 short pulses (10 s) followed by pauses (30 s) and was centrifuged at 10,000 × g for 20 min at 4°C. The supernatants were collected and loaded onto the column, which had been equilibrated in washing buffer. The eluate was collected in 1 mL microcentrifuge tubes. After several washes, the proteins were eluted using 8 mL of elution buffer (containing 150 mM imidazole). Enzyme activity was measured using a G6PD activity colorimetric assay kit (BioVision, catalog No. K757). The amount of NADH that was generated between time points was quantified at OD_450nm. One unit (U) of G6PD activity was defined as the amount of enzyme that catalyzes the conversion of 1.0 μmol of glucose-6-phosphate to 6-phosphoglucono-δ-lactone and 1.0 μmol of NAD⁺ to NADH per min at 37°C.

The wild-type G6PD structure (PDBID: 2HB9) was retrieved from the RCSB Protein Data Bank was subjected to point mutation in silico using the UCSF Chimera Rotamer tool [25]. Valine 291 was changed to methionine (V291M; G6PD_Viangchan). The side chain rotamer of the mutated residue was selected based on the Dynameomics rotamers library [26].

The G6PD_Viangchan variant was subjected to energy minimization in silico to optimize the initial geometry of the protein structure by removing possible steric clashes. The energy minimization was performed using the Gromacs simulation package (version 4.6.7) [27].

To understand the impact of mutation on the molecular interaction between G6PD_Viangchan and the structural NADP⁺, flexible small molecule and rigid protein docking was performed using AutoDock (version 4.2) [28]. Initially, the wild-type G6PD PDB file was cleaned by removing the existing NADP⁺ coordinate lines to create an “apoform” wild-type G6PD protein PDB file to be docked with the NADP⁺ structure using docking. The nonpolar hydrogen atoms were merged and total Kollman and Gasteiger charge were added to the protein. We ensured that there were no nonbonded atoms in the protein. Kollman and Gasteiger partial charges were also assigned to the NADP⁺ and all torsions were allowed to rotate during docking. Residues of the structural NADP⁺ binding site were specified based on the details obtained from previous work [29]. A grid box was assigned around the selected active site to cover the entire protein with a dimension of 60 × 60 × 60. The Lamarckian Genetic Algorithm (LGA) option was set to 150 runs with default parameter settings. The best conformation with the lowest docking energy was selected from the docking search. The interaction of structural NADP⁺ and wild-type G6PD including hydrogen bonds and hydrophobic interaction was analyzed using LigPlot+ (version 2.1) [30]. The same docking simulation approach was used for G6PD_Viangchan.

Mammalian Gene Collection (MGC) clones are commercially available sequence-validated full-length protein-coding cDNA clones for human, mouse, and rat from Dharmacon (GE Healthcare). pOTB7, which was used as the PCR template, contains human G6PD cDNA (GenBank RNA accession No. NM_001042351.2). The full-length of the gene contains 2295 bp nucleotides that include 128 bp upstream of ORF and 620 bp downstream sequences after the stop codon. The full-length coding region is 1548 bp long and can be translated into G6PD isoform B (515 amino acids). The forward and reverse primers were designed to carry restriction enzymes NdeI and XhoI, respectively. In addition, the reverse primer was also designed to remove the stop codon of the gene, which includes the His-Tag sequence. The size of the amplicon is 1,545 bp and the PCR product was cloned into the pET26b expression vector. Plasmid DNA were isolated from positive transformation colonies. Digestion with restriction enzymes NdeI and XhoI produced both vector and insert of the correct size, indicating successful G6PD gene cloning (Figure 1). Both pET26b+G6PD (wild type and Viangchan) were transformed into E. coli BL21 (DE3) competent cells for protein expression, which was induced by 0.1 mM and 1 mM IPTG. IPTG (1 mM) produced protein expression is 2-fold higher than 0.1 mM (data not shown). It was observed from SDS-PAGE that the protein expression level increased equally when the cells were incubated at different temperatures (16 °C, 25 °C, and 37 °C) (data not shown). SDS-PAGE showed optimum soluble G6PD expression when induced by incubation with 1 mM IPTG at 25°C for 16 h. The purified protein was run on 12.5% SDS-PAGE to confirm its molecular mass. The molecular mass of the protein is 59 kDa (Figure 2), which is similar to the mass previously reported for G6PD [5]. Western blotting showed a distinct single band indicating immunore-activity of target proteins to anti-G6PD, anti-6 × His-Tag, and anti-β-actin antibodies (Figure 3). A total yield of 1.32 mg and 0.82 mg of protein were obtained for wild-type and G6PD_Viangchan, respectively, per 1 L of culture. Enzyme activity of 205.76 U for wild-type G6PD and 67.12 U for G6PD_Viangchan were detected. The total specific activity for wild-type G6PD was 155.88 U/mg whereas for G6PD_Viangchan it was 81.85 U/mg.

Figure 2

Figure 3

The genomic sequence variation from G>A results in an amino acid substitution of valine by methionine and the protein variation exists at amino acid position 291. The residue is located at alpha helix loop 11 and is highly exposed to solvent. The alpha strand is connected to the Rossmann fold, which is characterized by an alternating motif of β-strand-α-helix-β-strand secondary structures. The initial β-α-β fold is the most conserved segment of the Rossman fold, and is responsible for FAD, NAD⁺, and NADP⁺ binding [31, 32]. It is assumed that the mutation might disrupt the orientation of the fold indirectly. A simple docking by using AutoDock revealed that the variant residues do not alter the NADP⁺ ligand binding site or the G6PD directly, but is located nearby. The binding energy obtained for NADP⁺ toward wild-type G6PD was –7.7 kJ/mol and that for G6PD_Viangchan was –7.6 kJ/mol. The NADP⁺ ligand is located at the center of the Rossman fold (Figure 4). The wild-type structure interacts with more amino acids than the variant structure, which results in the reduced protein production. Ligand–residue analysis by using LigPlot+ also shows the NADP⁺ interacts with almost 80% of the residues that exist within the Rossmann fold and indicates that the mutation might affect the orientation of the fold, resulting in reduced binding of the NADP⁺ ligand.

Figure 4