Histrap hp

All recombinant rVAR2 proteins were expressed in E. coli Shuffle cells (NEB) using a pET28 vector. The constructs of rVAR2 (ID1-ID2a) contained either a SpyTag [22 (link),23 (link)] or an albumin-binding domain (ABD) [24 (link)] for extension of plasma half-life fused in the N-terminal, and in the C-terminal a V5-tag and 6×His-tag were added for detection and affinity purification. The control proteins, DBL4 and DBL5, both contained a V5-tag and His-tag in the C-terminal similar to rVAR2 and for DBL5 an ABD was fused in the N-terminal.
Expression and purification of the rVAR2 protein were performed, as described in a previous publication [16 (link)]. Briefly, the E. coli pellet harboring the recombinant protein was resuspended in lysis buffer with benzonase and homogenized. After centrifugation and sterile-filtration, the supernatant was loaded onto a HisTrap HP (Cytiva, Uppsala, Sweden) column and eluted using imidazole. The protein was further purified by HiTrap SP HP (Cytiva, Uppsala, Sweden), before elution an endotoxin reduction wash step was included where 0.1% Triton X114 in binding buffer was passed over the column. After removing the wash buffer, protein was eluted with a linear gradient from 0 to 1 M NaCl in 25 mM phosphate buffer pH 7.2. The eluted rVAR2 protein was pooled and analyzed by SDS-PAGE, aliquoted, flash-frozen, and stored at −80 °C until use.

E. coli RNAP core and σ⁷⁰ were purified exactly as described previously (19 (link)). M. smegmatis and M. abscessus Arr were expressed in T7 express cells (New England Biolabs) transformed with pET28 expression vector encoding N-terminal 6×His-tagged M. smegmatis Arr or M. abscessus Arr. Expression was induced with 0.4 mM isopropyl-β-d-thiogalactopyranoside (IPTG) in exponentially growing cells, which were then incubated overnight at room temperature on an orbital shaker (150 rpm). Cells were then harvested by centrifugation and resuspended in grinding buffer (50 mm Tris-HCl [pH 7.9], 10% glycerol, 200 mM NaCl, and protease inhibitor mixture [Roche]). Cells were then lysed by sonication and debris cleared by centrifugation. Arr enzymes were then purified by HisTrap HP (Cytiva) nickel affinity chromatography, concentrated, and dialyzed into storage buffer (50 mM Tris-HCl [pH 7.9], 50% glycerol, 200 mM NaCl, and 2 mM β-mercaptoethanol).

The full length HSF5 expression construct was cloned into a pMAL-c6T vector (New England Biolabs) and fused to His⁶-MBP and a tobacco etch virus protease cleavage site at the N-terminus. His-MBP-full length HSF5 was purified by HisTrap HP (Cytiva), and gel filtration using a Superdex 200 pg 16/600 column (Cytiva). Size exclusion chromatography with multi-angle light scattering (SEC–MALS) was performed using a DAWN HELEOS8+ (Wyatt Technology Corporation, Santa Barbara, CA, USA), a high-performance liquid chromatography pump LC-20AD (Shimadzu, Kyoto, Japan), a refractive index detector RID-20A (Shimadzu), and a UV–vis detector SPD-20A (Shimadzu), which were located downstream of the Shimadzu liquid chromatography system connected to a PROTEIN KW-803 gel filtration column (Cat. no. F6989103; Shodex, Tokyo, Japan). Differential RI (Shimadzu) downstream of MALS was used to determine the protein concentrations. The running buffer used contained 25 mM HEPES/KOH (pH 7.2) and 150 mM KCl. Approximately 100 μL of the sample was injected at a flow rate of 1.0 mL /min. Data was then analyzed using ASTRA version 7.0.1 (Wyatt Technology Corporation). Molar mass analysis was also performed over half of the width of the UV peak top height. 30 min after incubation at 42 °C, 100 μL of MBP-full length HSF5 (12.6 μM) or HSF5-DBD (26.6 μM) was injected.

The preparations of recombinant human SOD1 and its mutants were previously described^{36 (link)}. Briefly, SOD1 with an N-terminal hexahistidine tag and a thrombin cleavage site was overexpressed in Escherichia coli BL21(DE3) strain (Agilent). The proteins were purified by Ni–NTA affinity chromatography (Ni Sepharose 6 Fast Flow, Cytiva). Zn ions were added to the obtained solutions, and the proteins were further purified by salting out. The purified protein solutions were treated with thrombin (Cytiva) to remove the hexahistidine tag, and then SOD1 without the hexahistidine tag was isolated by Ni–NTA chromatography (HisTrap HP, Cytiva). The protein solutions were finally dialyzed against acetate buffer containing ethylenediaminetetraacetic acid (EDTA) to obtain the apo form of SOD1 and its mutants. The sample concentration was adjusted to 20 μM in monomer units unless stated otherwise.

The pET-22b ( +)-CTX^WT and CTX^VAR plasmids were extracted from glycerol stock of DH10B cells using GF-1 Plasmid DNA Extraction kit (Vivantis, Malaysia) following the manufacturer’s protocol. The CTX^WT and CTX^VAR genes were amplified by touch-down PCR with their respective forward and reverse primers (Supplementary File 1-Table S2).
To express CTX^WT and CTX^VAR, pET-22b ( +) plasmids harboring the respective genes were first transcribed into RNA using the HiScribe™ T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, United States). The transcribed RNAs were then expressed using NEBExpress® Cell-free E. coli Protein Synthesis System (New England Biolabs, United States) for in vitro protein translation, according to the manufacturer's protocol. After that, CTX^WT and CTX^VAR were purified using a HisTrap™ HP, 1 mL affinity column on ÄKTA Pure protein purification system (Cytiva, United States). The HisTrap column was equilibrated with buffer A (20 mM sodium phosphate, 0.5 M NaCl, pH 8.0) before the samples were injected into the column. CTX^WT and CTX^VAR were purified with a gradient elution from 4 to 100% of buffer B (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 8.0) at a flow rate of 500 uL/min. The presence of his-tagged expressed CTX and its purity were visualized by western blot.

Biotinylated RNAP was purified from E. coli BL21DE3 cells transformed with pIA497 plasmid expressing RNAP β’ subunit fused with biotin carboxyl carrier protein, as described previously (Metelev et al., 2017 (link); Sabantsev et al., 2022 (link)). Recombinant GreA and GreB proteins carrying C-terminal 6xHis tag were expressed in E. coli Rosetta cells (NEB) transformed with pJL-GreA-CPH and pJL-GreB-CPH plasmids, respectively, and purified by Ni-chelating HisTrap HP (Cytivia) and gel-filtration Superdex 75 10/30 (Cytivia) column chromatography as described (Borukhov and Goldfarb, 1996 (link)).