Recombinant Expression and Purification of NadA

A gene fragment of NadA (GenBank–NP_274986) encoding globular head domain (A²⁴ to G⁸⁵) and first domain of coiled-coil region (L⁸⁶ to A¹⁷⁰, details of the sequence are in Figure 1) was amplified from genomic DNA isolated from NM (Strain MC58, isolate–M1/03). Details on primers (NadA F and NadA R primers) and amplicon length are presented in Table 1. PCR product was digested with BamHI and KpnI enzymes and ligated into in-house modified pQE-30-mCherry-stop-GFP plasmid as described earlier (Mertinková et al., 2020 (link)). The cloned vector was electroporated into E. coli M15 strain (Qiagen, Germany). Clonal selection on LB-carbenicillin agar plate (LB broth, Sigma Aldrich, Germany, carbenicillin, 50 μg/mL, Duchefa Biochemie BV, Haarlem, The Netherlands), overexpression of the recombinant protein, its purification with nickel affinity chromatography (Ni-NTA agarose beads, ABT, Spain) followed by anion exchange [Bis-Tris, pH 6.0 containing 8M urea (Sigma Aldrich) for binding; Bis-Tris, pH 6.0 with NaCl (Sigma Aldrich) gradient for elution] was performed as described in our earlier publications (Jiménez-Munguía et al., 2018 (link); Kánová et al., 2019 (link)). The purified protein was stored in 10 % glycerol (MikroChem spol. SRO, Pezinok, Slovakia) at −20°C in several aliquots.