Hitrap sp hp column

The generation of recombinant human BPI variants was performed as described [40 (link)] with slight modifications. In brief, a pCR3 vector (Invivogen, Toulouse, France) construct comprising an N-terminal HA signal peptide, amino acids 32 to 487 of either BPI_216E or BPI_216K, and a C-terminal FLAG-Tag was transfected in Expi293F™ cells using the ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific, Waltham, MA, USA). The expressed protein was purified by cation exchange chromatography via a HiTrap™ SP HP column (Cytiva, Marlborough, MA, USA). Fractions containing the protein of interest were pooled and purified by size exclusion chromatography using a HighLoad 16/600 Superdex 75 pg column (Cytiva, Marlborough, MA, USA), concentrated via ultrafiltration (Amicon Ultra-15, Merck Millipore, Darmstadt, Germany), and dialyzed against PBS. Concentration was determined by DC-Protein Assays (Bio-Rad Laboratories, Feldkirchen, Germany).

PTN and its domains were produced following previously reported protocols (Feng et al., 2021 (link)). Briefly, the mature PTN open reading frame was cloned into a pET-15b vector using NcoI and XhoI restriction sites and transformed into OrigamiB(DE3) cells. The transformed cells were grown under conditions similar to the Q163C/Q309C mutant of the α_MI-domain. To extract the protein, cell pellets were resuspended in 20 mM Tris, 0.2 M NaCl, pH 8.0, 1 mg/mL of lysozyme and incubated for 20 minutes at room temperature. After sonication and centrifugation to remove insoluble material, the protein is extracted from the supernatant using a 5-mL HiTrap SP HP column (Cytiva) and eluted with a 0.1–1.5 M NaCl gradient. To produce PTN domains, a pHUE vector(Catanzariti et al., 2004 (link)) containing PTN ORF coding either residues G1 to C57 (PTN-NTD), residues N58 to K114 (PTN-CTD Δtail), or residues N58 to D136 (PTN-CTD) at the 3’ end of the ubiquitin ORF was transformed into OrigamiB(DE3) cells. Cells were grown in M9 media at 37 °C to an OD₆₀₀ of 0.8. 0.25 mM IPTG was added to the culture and incubated overnight at 23°C. Purifications of PTN domains and various mutants were similar to that of α_MI-domain except no size exclusion chromatography was used.

The antibodies in THIOMAB format with S400C mutated cysteine were expressed in Expi293 or CHO cell lines, and purified by MabSelect SuRe (Cytiva) followed by size exclusion or SP cation exchange chromatography. The purified antibodies were adjusted to pH 8.0 using 1 M Tris pH 8.5, and supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA), and 50-fold molar excess of dithiothreitol (DTT) to reduce the blocked cysteine residues. After LC-MS confirmation of complete blocker removal, the antibodies were diluted 10 times with Buffer A (10 mM Succinate, pH 5.0), and loaded onto a 5 ml HiTrap SP HP column. The column was then washed with 10 column volumes of Buffer A, and eluted with 50 mM Tris, 150 mM NaCl, pH 8.0. The purified reduced antibodies were then re-oxidized with 15-fold molar excess of dehydroascorbic acid (DHAA) and 2 mM EDTA. The samples were then loaded onto a 5 ml HiTrap SP HP column (Cytiva) and washed with 10 column volumes of Buffer A. The antibodies were then gradient eluted with 30–100% Buffer B (10 mM Succinate, 300mM NaCl, pH 5.0) in 20 column volumes.

E. coli Shuffle T7 (DE3) cells harboring the protein gene of interest were grown in LB at 37 °C to OD₆₀₀ 0.6–0.8, induced with 0.1 mM β-d-1-isopropyl thiogalactopyranoside and incubated further for 24 h at 18 °C. Cells were pelleted and stored at −80 °C before protein purification. The cells were disrupted by sonication in a 20 mM Tris-HCl buffer, pH 8.0. The sonicated extract was separated into soluble and insoluble fractions by centrifugation at 12,000 × g for 15 min at 4 °C. The soluble fraction was dialyzed against 10 mM sodium acetate buffer, pH 5.0, and filtered before applying to a RESOURCE Q column (6 mL, Cytiva) or HiTrap SP HP column (5 mL, Cytiva) previously equilibrated with the same buffer. The elution was done with a linear gradient of NaCl from 0 to 0.3 M in the same buffer. The recombinant protein fractions were collected and dialyzed against a 5 mM Tris-HCl buffer containing 150 mM NaCl, pH 8.0. Purified recombinant proteins were concentrated using a Millipore centrifugal protein concentration device (10 kDa cutoff) and loaded onto a Superdex200 Hiload 16/600 column (Cytiva) equilibrated with 5 mM Tris-HCl buffer containing 150 mM NaCl, pH 8.0. Protein purity was confirmed by SDS–PAGE, and protein concentrations were measured spectrophotometrically using molar absorption coefficients calculated in ProtParam (http://expasy.org/tools/protparam.html).

The YSK₂ and YSK₂ constructs were expressed and purified using a slight modification of Livernois et al. [39 (link)]. Briefly, the cells were lysed by boiling for 20 min with agitation every 5 min and were subsequently cooled on ice. Sodium acetate (pH 5.0) was added to a final concentration of 20 mM and the supernatant was filtered before continuing with purification. The filtered supernatant was loaded onto a 5 mL HiTrap SP HP column (Cytiva, Vancouver, BC, Canada) equilibrated with 20 mM sodium acetate, pH 5.0 (buffer A). Elution occurred over a gradient of 0–1 M NaCl (Buffer B: 20 mM sodium acetate, pH 5.0, 1 M NaCl). Fractions containing the protein of interest were desalted into Milli-Q water using a HiPrep 26/10 column (Cytiva). After concentrating the eluted protein fraction, reversed-phase HPLC was performed. The protein sample was purified using a BioBasic C4 column (Thermo Scientific, Ottawa, ON, Canada), which was equilibrated with Buffer A (0.1% trifluoroacetic acid (TFA) (w/v)). The protein was eluted using a 1–100% Buffer B (0.1% TFA (w/v) in acetonitrile) gradient. Sample purity was determined using 12% SDS-PAGE and the fractions containing the protein of interest were pooled, flash frozen and lyophilized. The proteins were stored at −20 °C until use.

Endolysin expression vectors were introduced into E. coli BL21 (DE3) Star (Invitrogen, United States) for the expression of protein. Plasmid harboring gene encoding CecA-fused EGFP was introduced into E. coli BL21 (DE3) pLysS (Novagen) for expression. The cells were grown in LB broth to an exponential phase (OD₆₀₀ = 0.4–0.5) and then induced with isopropyl β-D-1-thiogalactopyranoside (IPTG; Duchefa, Netherlands) at a concentration of 0.5 mM at 25°C for 5 h. The bacterial cells were harvested by centrifugation and the pellets washed with phosphate-buffered saline (PBS). The cells were resuspended in lysis buffer (20 mM Tris–HCl, pH 7.5, 300 mM NaCl, and 20 mM imidazole) and disrupted by sonication (Sonics, United States) for 5 min. The supernatant was obtained by centrifugation at 15,000 × g for 30 min from cell lysate. The extracts were loaded onto an Ni-NTA affinity chromatography column using FPLC (AKTA go, Cytiva, United Kingdom). The proteins were eluted using a linear gradient from 20 to 500 mM of imidazole. The proteins were then loaded onto a HiTrap SP HP column (Cytiva) for cation exchange chromatography and eluted with a linear gradient of NaCl from 0 to 1 M in 20 mM Tris–HCl, pH 7.5. The proteins were then dialyzed in the storage buffer [20 mM Tris–HCl, pH 7.5, 150 mM NaCl buffer (pH 7.5)].