Construction and expression of recombinant proteins
StpA and GrpE recombinant proteins were purified in E. coli using the pETite C-His vector utilizing the Expresso T7 system protocol (Lucigen) as described in Brandt et al. (60). Genes were amplified by PCR from B. burgdorferi strain B31-5A4 genomic DNA using primers listed in Table S2. pETite C-His plasmids were transformed into E. coli Top 10G Solo cells (Lucigen) and plated on LB plates supplemented with 50 μg/ml kanamycin. Positive transformants were screened by PCR and confirmed by sequencing. Correct plasmids were transformed into E. coli BL21(DE3) cells (Lucigen). Positive transformants were screened and grown in LB-kanamycin (50 μg/ml) overnight. The overnight culture was used as an inoculum at a 1:100 dilution in LB-kanamycin (50 μg/ml) in 100 ml for protein expression. Protein expression was induced by the addition of isopropyl-d-thiogalactopyranoside (IPTG; 1 mM) when cells were at mid-logarithmic phase (OD600 = 0.4-0.6). Cells were harvested at late-log-phase growth (3-4 h), and recombinant protein was purified under nondenaturing conditions using a nickel-nitrilotriacetic acid (Ni-NTA) Fast Start His tag affinity purification kit (Qiagen) per the manufacturer’s protocol. Proteins were dialyzed into PBS (pH 7.4) and quantified by bicinchoninic acid (BCA) assay (Thermo-Fisher Scientific). Protein purification was examined by immunoblot and Coomassie brilliant blue staining.
plzA R145D and R145K mutant alleles were generated using splice overlap exchange techniques (Table S2) (53,61). The amplicons were digested with AgeI and XhoI, ligated into pET45b(+) using Instant Sticky-end Ligase Master Mix (New England Biolabs), and purified using a QIAquick PCR Purification Kit (Qiagen). plzA R145D F92A F95A mutant and plzA R145D F168A F218A mutant gene sequences were synthesized with flanking BamHI and EagI restriction sites and provided in pET45b(+) for expression with an N -terminal Hexa-His-tag (GenScript). To propagate the plasmids, E. coliNovaBlue(DE3) cells (Novagen) were transformed according to the manufacturer’s protocol with the following modification: 100 ng of the plasmid was added to 20 µl of competent cells. Transformants were selected with 100 µg/ml ampicillin. Positive transformants were screened by PCR and confirmed by DNA sequencing. Isolated colonies were selected and transferred into 10 ml of prewarmed LB-ampicillin (100 μg/ml) and grown overnight (37°C; 250 rpm). The overnight culture was used as an inoculum at a 1:200 dilution in LB-ampicillin (100 μg/ml) in 500 ml for protein expression. Protein expression was induced by the addition of 1 mM IPTG when cells were at mid-logarithmic phase (OD600= 0.4). Cells were harvested at late-log-phase growth (4 h) (5000 ×g ; 15 min; 4°C). The cell pellet was resuspended and vortexed in binding/washing (B/W) buffer (20 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, pH 7.4) containing protease inhibitor cocktail (1% v/v; Sigma) and Pierce Universal Nuclease for Cell Lysis (ThermoFisher; final concentration 0.5 μg/ml). The samples were passed through an EmulsiFlex-C3 high-pressure homogenizer (three times at 1000-1500 psi; 4°C), and the resulting slurry was centrifuged (15,500 × g ; 30 min; 4°C) to separate the soluble and insoluble fractions. To purify proteins from the soluble fraction, nickel affinity chromatography was performed using a Fast Protein Liquid Chromatography platform (ÄKTA Pure 25 M; Cytiva) with a 1-ml HisTrap FF column (Cytiva) at a flow rate of 1 ml/min (4°C). The column was equilibrated with five column volumes of B/W buffer. Samples were loaded into a 10 ml Superloop (Cytiva), followed by washing with 20 column volumes of B/W buffer. Proteins were eluted with five column volumes of elution buffer (20 mM NaH2PO4, 500 mM NaCl, 250 mM imidazole). One-ml fractions were collected from under the 280 nm peak, combined, and dialyzed into phosphate-buffered saline (PBS, pH 7.4) overnight at 4°C using Spectra/Por 1 (6–8 kDa cutoff) dialysis membranes (Spectrum Laboratories). The concentrations of the recombinant proteins were determined using a BCA assay (Pierce) as instructed by the supplier.