4.2. cDNA Cloning, Recombinant Protein Expression, and Metal Ion Affinity Purification

The cloning of the nxh1 nucleotide sequence (EMBL accession number: {"type":"entrez-nucleotide","attrs":{"text":"AF197563","term_id":"6467951","term_text":"AF197563"}}AF197563), as well as the expression and purification of recombinant NXH1 (rNXH1), were carried out as previously described [76]. The procedures described below refer specifically to NXH8.

The nxh8 (EMBL accession number: {"type":"entrez-nucleotide","attrs":{"text":"AJ344067","term_id":"15144253","term_text":"AJ344067"}}AJ344067) nucleotide sequence was obtained from a phage cDNA library [74,75]. The nxh8 cDNA fragment was released from the phage DNA by EcoRI and NotI double digestion and was subcloned at the HincII site of pGEM3Zf(+) (Promega Corporation, Madison, WI, USA) by blunting the ends with DNA Polymerase I Large (Klenow). DNA sequencing confirmed that the nxh8 fragment had been successfully subcloned in a reverse orientation.

For expression, the nxh8 cDNA fragment was amplified by PCR and was subcloned at the BamHI site of the pRSET-C vector (Thermo Fisher Scientific, Waltham, MA, USA), which allows the expression of a 6x His-tagged recombinant protein at the N-terminus. This strategy used the M13 forward primer combined with a specific oligonucleotide (5’—cgg atc ctt gaa tgt aag ata tgc aac ttc—3′) that includes an upstream BamHI restriction site (in bold) in frame with the first nucleotides of the mature NXH8 peptide coding sequence. The correct orientations of the clones were evaluated by double BamHI/XbaI digestion and restriction analysis and were confirmed by DNA sequencing.

DNA sequencing was performed using the chain-terminator method [121]. The reactions were run in a PTC-100 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) using a BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. After purification, DNA sequence analysis was performed using an ABI Prism 3100 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA).

Chemically competent E. coli BL21 (DE3) cells were transformed with the pRSET-C-nxh8 plasmid using standard protocols [122]. Transformed cells were cultured at 37 °C, under constant agitation (200 rpm), in ampicillin (100 μg/mL)-supplemented Luria Broth (LB). Recombinant protein expression was induced by adding IPTG (1 mM) when the cell culture optical density at 600 nm (OD_600nm) reached 0.6.

After 3 h of induction, the cells were harvested by centrifugation (1000× g, 4 °C, 20 min) and resuspended in 50 mM Tris, pH 8.0, containing 100 mM NaCl, 10 mM EDTA, 1 mM PMSF, and 0.1% Triton-X 100. Cell lysis was achieved by sonication (3 × 1 min cycles, 60% amplitude, 20 kHz frequency, duty cycle of 0.5), with the samples kept in an ice bath during the process. Soluble and insoluble protein fractions were separated by centrifugation (12,000× g, 4 °C, 20 min).

Recombinant NHX8 (rNXH8) was expressed as inclusion bodies that were resuspended in 50 mM Tris (pH 8.0), containing 8 M urea, 100 mM NaCl, and 10 mM 2-mercaptoethanol (2-ME). The samples were incubated on a tube roller for 1 h at room temperature and subsequently centrifuged (10,000× g; 4 °C; 15 min). The resulting supernatant was reserved for the refolding process.

After solubilizing the inclusion bodies, the recovered recombinant proteins were renatured by dilution (1:200, v/v) in 100 mM Tris, pH 8.0, containing 150 mM NaCl, and 2 mM GSH-GSSG. The diluted protein solution was then kept under magnetic agitation for 10 h at 14 °C. Any protein aggregates were removed by centrifugation (12,000× g; 4 °C; 15 min) or by filtration through a 0.22 μm filter.

Renatured recombinant proteins were applied to a Ni²⁺-charged chelating resin for metal-ion chromatography (Cytiva, Marlborough, MA, USA). The resin was washed with five column volumes (cv) of 50 mM Tris, pH 6.3, containing 5 mM imidazole, and 100 mM NaCl. Protein elution was first achieved with 5 cv of 50 mM Tris, pH 8.0, containing 250 mM imidazole, and 100 mM NaCl, and then with 5 cv of 50 mM Tris, pH 8.0, containing 100 mM EDTA, and 100 mM NaCl.

Alternatively, rNXH8 was purified under denaturing conditions, with previously solubilized inclusion bodies being directly applied to a Ni²⁺-charged chelating resin for metal-ion chromatography. For this, the column was first equilibrated with 5 cv of 50 mM Tris, pH 8.0, containing 8 M urea, 100 mM NaCl, and 10 mM 2-ME, and washed with 5 cv of the wash buffer (see above) containing 4 M urea. Recombinant proteins were eluted with 5 cv of the elution buffer (see above) containing 2 M urea.

For recombinant proteins that were previously renatured, the eluted fractions were dialyzed against 10 volumes of phosphate-buffered saline (PBS) containing 5 mM EDTA, using semipermeable membranes with a nominal cutoff of 3500 Da. Four buffer changes were conducted at 4 °C at 12 h intervals. For recombinant proteins purified under denaturing conditions, the rNXH8 concentration was lowered to 0.1 mg/mL to reduce protein aggregation during dialysis against 10 volumes of PBS buffer containing 2 M urea, 2 mM GSH-GSSG, 10 mM 2-ME, and 5 mM EDTA. After 12 h of dialysis at 4 °C, 50% of the buffer was replaced with PBS containing 5 mM EDTA, resulting in four successive buffer changes.