Hitrap chelating column

Human MSH2 and His-tagged MSH3 were overexpressed in SF9-insect cells using a pFastBac dual-expression system (GIBCO-BRL), and purified as follows11 (link)30 (link). MSH2 and his-tagged MSH3 were overexpressed in SF9-insect cells using a pFastBac dual-expression system. Briefly, the supernatant was loaded onto a 5 ml HiTrap chelating column (GE Healthcare) charged with Ni-NTA affinity column and equilibrated with lysis buffer. The bound proteins were then eluted with a 25 ml 20–200 mM imidazole gradient. The peak fractions containing the MSH2/MSH3 (eluted at 140 mM imidazole) and were then loaded onto a Mono P and HiTrap Heparin column (GE Healthcare) connected in tandem and equilibrated in column buffer (25 mM HEPES NaOH, pH 8.1, 0.1 mM EDTA, 10% glycerol (v/v) and 1 mM DTT) containing 300 mM NaCl. The MSH2/MSH3 containing fractions were then applied to MonoQ (GE Healthcare). MSH2/MSH3 fractions were stored in 20% glycerol (v/v), aliquoted and frozen at −80 °C.

Recombinant vWbp was isolated as reported^{51 (link)}. Recombinant vWF-A1 domain was produced in E. coli M15 cells harboring the plasmid pREP4 (Qiagen), originating from plasmid pQE30-vWF-A1 as previously reported^{52 (link),53 (link)}. The A1 domain contained an N-terminal His tag and was purified by Ni²⁺-affinity chromatography on a HiTrap chelating column (GE Healthcare, Buckinghamshire, UK).

AmrZ and FleQ were purified as described before (Prada-Ramírez et al., 2016 (link); Pérez-Mendoza et al., 2019 (link)). For FleN purification, the One Shot BL21star (DE3) (pET28b(+):fleN) cells were grown at 28°C in 2-L Erlenmeyer flasks containing 1 L of 2 × YT culture medium (Sambrook et al., 1989 ) supplemented with kanamycin (50 μg/ml). Protein expression was induced at an A₆₆₀ of 0.2–0.3 by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside and cultures were grown for another 5 h at 15°C, when they were harvested by centrifugation at 5000 × g. The pellet resulting from a 500 ml culture was resuspended in 25 ml of buffer A (25 mM Na-phosphate pH 7.0, 500 mM NaCl, 5% glycerol) with protease inhibitor mixture (Complete™, Roche) and broken by treatment with 20 μg/ml of lysozyme and French press. Following centrifugation at 13,000 × g for 60 min, the FleN protein was predominantly present in the soluble fraction. The supernatant was loaded onto a 5 ml Hi-Trap chelating column (GE Healthcare), equilibrated with buffer A and eluted with a gradient of a 50 mM-1 M imidazole. Fractions containing His₆-FleN were pooled and was dialyzed against 20 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol and stored at −80°C. Protein concentrations were determined using the Bio-Rad Protein Assay kit.

The His-tagged proteins expressed in each strain were purified from the respective crude membrane extracts (CMs) as follows. A 5 ml Hitrap chelating column (GE Healthcare) was charged with nickel and equilibrated with 10 column volumes of buffer A (0.1 M sodium phosphate buffer, pH 8.0, 500 mM NaCl, 8 M urea). The protein sample was filtered through a 0.45 µm filter and loaded onto the column. Once the OD₂₈₀ returned to baseline the column was washed with buffer B (0.1 M sodium phosphate, pH 6.3, 500 mM NaCl, 8 M urea). The bound protein was eluted using a linear gradient of 15 column volumes to 50% buffer C (0.1 M sodium phosphate, pH 6.3, 500 mM NaCl, 8 M urea, 500 mM Imidazole) and a step to 100% buffer C which was maintained for 5 column volumes.

E. coli C43 (DE3) harboring plasmid encoding VcaM was cultivated at 37 °C, induced with 0.2 mM IPTG and overexpressed at 24 °C. The cells were collected by centrifugation and disrupted by using French press. The pellet was removed by centrifugation, and the supernatant collected. The supernatant was centrifuged at 43,000 rpm for 60 min to spin down the cell membranes, which were collected and solubilised in 2% (v/v) Triton X-100 (BioShop, Burlington, ON, Canada). Detergent solubilized VcaM was purified by using nickel affinity column (Hitrap chelating column, GE, Boston, MA, USA) [59 (link)].