T7 express e coli cells

Human CLIC5 (Uniprot Q9NZA1; CLIC domain (residues 16-251; termed CLIC5)), human CLIC2 (Uniprot O15247; residues 2-247), mouse CLIC6 (Uniprot Q8BHB9; residues 363-596), EXC-4 (Uniprot Q8WQA4; residues 2-290), rat 14-3-3ε (Uniprot P62260) and 14-3-3θ (Uniprot P68255) were sub-cloned into a pETM11 vector. A GAMG cloning artifact sequence, originating from the remanent TEV cleavage site and a NcoI restriction site, remained at the amino terminus. The F34D mutation was introduced using the standard QuickChange approach, and deletion of residues 57-68 was achieved by using the NEBaseChanger® workflow. The rat DOC2B (a kind gift from Prof. Daniel Khananshvili, Tel Aviv University; Uniprot P70610; residues 117-412) was sub-cloned into pRSFduet vector. All constructs were verified by sequencing. All proteins were expressed in T7 Express E. coli cells (New England Biolabs) as previously described in ref. ^{13 (link)}. Briefly, bacterial cultures were grown to mid-log phase in terrific broth (Formedium) at 37 °C and induced with 0.3 mM isopropyl β-D-1-thiogalactopyranoside (Formedium) overnight at 16 °C. Cells were harvested by centrifugation at 5500 x g for 15 min at 4 °C, and cell pellets were stored at −80 °C until use.

The full-length KaPOx gene was synthesized with a C-terminal 6×His tag and inserted into the pET-21b(+) expression vector, in which the standard N-terminal T7-tag was excluded (BioCat). This plasmid was then transformed into chemically competent E. coli T7 Express cells (New England BioLabs) according to the standard 5-min transformation protocol. Sequencing (Microsynth) confirmed the identity of the plasmid. The cultivation of E. coli cultures was carried out routinely in terrific broth (TB) Amp⁺ buffered at pH 7.5 and supplemented with ampicillin (100 μg ml⁻¹) at 37°C. Cultures were incubated at 20°C for 20 h in the presence of 1.0% (wt/wt) lactose to induce expression of KaPOx. Cell disruption and immobilized metal affinity chromatography were carried out as previously described (61 (link)), with the adaptation of using 50 mM Tris-HCl based buffers at pH 8.0. Active fractions were pooled and dialyzed at 4°C against 50 mM potassium phosphate buffer (PPB; pH 6.5) using 7-kDa-cutoff Membra-Cel (Serva) dialysis tubing. After dialysis, the yellow KaPOx precipitate was harvested from the tube and washed twice with 50 mM PPB (pH 6.5) by centrifugation at 1,000 × g and 4°C for 120 s. Homogeneity of the purified protein was confirmed by SDS-PAGE and LC-ESI-MS peptide mapping.

yonO was cloned into the pET-28a expression vector, and YonO was expressed in E. coli T7 Express cells (New England Biolabs) with an N-terminal 6xHis-tag. To obtain YonO^3D>3N, the yonO pET-28a construct was subjected to site-directed mutagenesis using Quikchange XL II (Agilent). Cells were grown in LB at 37 °C, and protein expression was induced at OD₆₀₀ of 0.4 with 1 mM isopropyl-β-D-1-thiogalactoside (IPTG) at 18 °C for 16 h. Harvested cells were re-suspended in grinding buffer (50 mM Tris-HCl pH 7.9, 200 mM NaCl, EDTA-free protease inhibitor cocktail (Roche)) and disrupted by sonication. The lysate was clarified by centrifugation and made to 20 mM imidazole before being loaded on a His Trap HP column (GE Healthcare) pre-equilibrated with 20 mM Tris-HCl pH 7.9 and 600 mM NaCl. Protein eluted in 100 mM imidazole was bound to a HiTrap Heparin column (GE Healthcare) equilibrated in 10 mM Tris-HCl pH 7.9 and 600 mM NaCl, and eluted by a gradient increase of NaCl concentration to 1 M. Fractions containing YonO were concentrated and further purified on a Superdex 200 16/60 column equilibrated in 50 mM Tris-HCl pH 7.9 and 500 mM NaCl. Purified YonO was concentrated and dialysed overnight into storage buffer (20 mM Tris-HCl pH 7.9, 50% glycerol, 200 mM KCl, 1 mM dithiothreitol and 0.1 mM EDTA) and stored at −20 °C.

The pET-28a Expression vectors were obtained from Novagen Inc. (Sigma Merck, Johannesburg, South Africa), E. coli T7 Express cells obtained from New England Biolab (Inqaba Biotec, Pretoria, South Africa). NMN, ATP, NAD, GTP, alcohol dehydrogenase (ADH), ANS (8-anilino-1-naphthalene sulfonic acid), mant-ATP, and SYPRO Orange were purchased from Sigma Merck (Johannesburg, South Africa). Other biochemicals and reagents used were of analytical grade.

MSP1E3D1 was synthesized in E. coli T7express cells (New England Biolabs, Frankfurt, Germany) and purified by taking advantage of a terminal His-tag^{63 (link)}. For removal of the N-terminal His₆-tag, the protein was TEV digested followed by a reverse IMAC. The MSP1E3D1 solution was set to 1 mM DTT before TEV was added in a MSP1E3D1 to TEV ratio of 1:25. The mixture was dialyzed against 1 mM DTT, 0.5 mM EDTA, 50 mM Tris-HCl, pH 8.0 at 4 °C over night. Before loading on a pre-equilibrated HiTrap™ IMAC FF column (Cytiva, Munich, Germany), the mixture was centrifuged at 20,000 × g and 4 °C for 10 min. The flow through was collected and the column was washed with 10 column volumes (CVs) equilibration buffer (20 mM IMD, 100 mM NaCl, 20 mM Tris-HCl, pH 8.0). The flow through and wash fractions were concentrated to 3–5 mg/mL using Amicon ultrafiltrators (10 kDa MWCO, Merck Millipore, Darmstadt, Germany). All fractions were combined and dialyzed 2× over night at 4 °C against 5 L 10% (v/v) glycerol, 300 mM NaCl, 40 mM Tris-HCl, pH 8.0. Until ND formation, the protein was flash frozen in liquid nitrogen and stored at −80 °C.