Histrap ff column

BL21 (DE3) cells from a single colony, containing the chosen protein expression plasmid, were grown in LB overnight then sub-cultured in 2×YT medium until OD₆₀₀ reached 0.6. For pBAD and pRSFDuet plasmids, protein expression was induced by adding arabinose or isopropyl ß-D-1-thiogalactopyranoside (IPTG), respectively. Cells were harvested 2–3 hr later and lysed using a cell disrupter (Constant Systems Ltd.). Proteins were purified from inclusion bodies using Nickel affinity chromatography on prepacked HisTrap FF columns (Cytiva, UK), followed by ion exchange chromatography on either HiTrap Q HP or HiTrap SP HP columns (Cytiva, UK) depending on protein charge, described in full previously (Pereira et al., 2019 (link)). Proteins from pE-SUMOpro plasmids (those containing DHFR domains) were expressed by adding IPTG, and cells harvested after 18 hr of further growth at 18°C. Proteins were purified at 4°C from the soluble fraction, essentially as before (van Loon and Schatz, 1987 (link)), but with 250 mM NaCl in their ‘Buffer C’. A further purification step on a HiLoad 16/60 Superdex gel filtration column (Cytiva, UK) was included to remove remaining contaminants. A full list of PCPs, their amino acid sequences, and respective expression vectors are given in Supplementary file 1.

The pL polyprotein plasmids were transformed into BL21 (DE3) pLysS E.coli cells. 2 ml LB starter culture was used to inoculate 0.5 L autoinduction medium^{69 (link)}. Totally, 10 × 0.5 L cell cultures were incubated at 28 °C, 200 rpm for 24 h, for protein expression and then harvested. Cell pellets were resuspended in lysis buffer (20 mM Tris, 300 mM NaCl, 10 mM Imidazole, 2 mM Benzamidine, 1 mM PMSF, 0.01% Triton-X-100, pH 8), passed through a cell disruptor at 30 kPsi, 4 °C and then centrifuged at 25,000 rpm, 25 mins, 4 °C. The supernatant containing overexpressed protein L constructs was loaded onto 2 × 5 ml HisTrap FF columns (Cytiva) equilibrated with wash buffer (20 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 8). Unbound protein was eluted with wash buffer before pL was eluted with 100% elution buffer (20 mM Tris, 300 mM NaCl, 500 mM imidazole, pH 8). Protein was further purified using a 5 ml HiTrap Q HP anion exchange chromatography column (Cytiva) for ion exchange purification. A gradient elution of 0–100% elution buffer (25 mM sodium phosphate pH 7.4, 0–500 mM NaCl) was performed over 300 ml. Protein was finally purified by size exclusion chromatography (HiLoad 26/60 Superdex 75 pg, Cytiva) and eluted in 25 mM sodium phosphate, pH 7.4. The purified protein was dialysed into ultra-pure water (Milli-Q, resistivity ≥18.2 MΩ cm) and lyophilised for storage at −20 °C.