Ni nta affinity chromatography column

After amplification using primers PmrAF and PmrAR, PCR products were inserted into pET-28a(+) digested with NdeI and HindIII to add a carboxyl terminal 6-his-tag. Plasmids were transformed into the expression strain E. coli BL21 (DE3) and cultured in LB medium (5 ml) at 37°C overnight. Cells were diluted 1:100 into 1 L LB broth containing ampicillin and cultured. When OD₆₀₀ was between 0.4 and 0.5, recombinant PmrA was activated with 0.1 mM IPTG at 16°C for 20 h.
Mixtures were centrifuged at 7,000 × g for 20 min at 4°C and resuspended in lysis solution, followed by harvest of 6 L of cells and disruption by a One Shot Cell Disrupter (Constant Systems, Daventry, UK). To isolate insoluble substances, mixtures were passed through 0.45-μm syringe-end filters after centrifuging at 12,000 × g for 20 min at 4°C before loading on a His-tag column. Supernatants were transferred to a Ni-NTA affinity chromatography column (GE Healthcare, Pittsburgh, PA, USA). After balancing the column with lysis buffer, recombinant protein was added and eluted using washing buffer with an imidazole concentration gradient (0, 30, 60, 100, 200, and 500 mM). Washing buffer (30 ml) containing target recombinant proteins was dialyzed in 20 mM Tris-HCl, pH 8.0. Proteins were subjected to SDS-PAGE. Unstained and prestained protein markers (Fermentas, SM0431 and SM0671 Waltham, USA) were applied as references.

The recombinant strains were collected by centrifugation at 6000× g for 20 min and resuspended in a lysis buffer (50 mM Tris-HCl, pH 7.1). The cells were then sonicated and centrifuged at 14,000× g for 30 min at 4 °C to remove the debris. The supernatants containing the target proteins were loaded onto a Ni-NTA affinity chromatography column (GE Healthcare) and purified using a 20–100 mM imidazole gradient. The purified enzyme was dialyzed against 50 mM phosphate-citrate buffer (pH 5.4) and concentrated to 1.5 mg/mL. The protein homogeneity was confirmed by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

The pET28a-6xHis-TEV-LbCas12a-NLS-3xHA plasmid was transformed into the Rosetta (DE3) E. coli strain and protein was induced with 500 μM IPTG at 18 °C for 18 h. Cells were collected and lysed, followed by Ni-NTA affinity chromatography column (GE Healthcare) and Superdex 200 10/300 column (GE Healthcare). The purified proteins were concentrated with Amicon ultra-4 centrifugal filters-50K (Merck Millipore) and quantitated with BCA protein assay Kit (Thermo Fisher Scientific). The proteins were stored at −80 °C.

Genes encoding Prdx6 and Trx were cloned from toad B. maxima skin cDNA into the pET-28a (+) vector with an N-terminal His₆-Tag. rePrdx6 ^{(C46A, C90A)} and reTrx ^{(C32A, C35A)} mutants were cloned by point-mutation PCR into the pET-28a (+) expression vector with an N-terminal His₆-Tag. Competent E. coli strain Rosetta (DE3) (TsingKe) was transformed with the recombinant plasmids.
Transformed colonies were grown to an A₆₀₀ of 0.8 in a shaking incubator (200 rpm, 37 °C) and then treated with 2 mM IPTG to induce target protein expression for 20 h in a shaking incubator (200 rpm, 16 °C). E. coli pellets was resuspended in precooled equilibration buffer (50 mM Tris–HCl and 0.5 M NaCl, pH 7.5) for ultrasonication. Supernatants were collected by centrifuging at 16,000g for 40 min. The supernatant was loaded onto a pre-equilibrated Ni-NTA affinity chromatography column (GE healthcare). Various concentrations of imidazole (equilibration buffer containing 10, 50, and 80 mM imidazole) were applied to wash extensively. Columns were eluted by equilibration buffer containing 300 mM imidazole. Purified recombinant proteins were dialyzed and concentrated in PBS for hemolysis assays.

The lipase was purified with protocol described in detail previously [19 (link)]. The bacterial pellets were resuspended in 15 ml lysis buffer (50 mM Tris-Cl pH 8.0), disrupted by sonication, followed by centrifugation at 10,000 × g for 30 min at 4°C. The concentrated supernatant (crude enzyme) was applied to the Ni-NTA affinity chromatography column (GE Healthcare Life Sciences, Piscataway, USA) which was equilibrated with washing buffer (50mMTris-ClpH8.0, 500 mM NaCl, and 10% glycerol). The enzyme protein was eluted using an imidazole gradient (0, 20, 40, 80, 100, and 200 mM) in washing buffer. After the elution, the imidazoles in the purified enzyme were removed by dialysis in 50 Mm Tris-Cl (pH 8.0). The purified enzyme was analyzed by SDS-PAGE. The single protein band after purification was confirmed by the peptide mass fingerprinting.

The bacterium containing recombinant Cel-5A was cultured in LB liquid medium supplemented with 100 μg/mL Kan on a rotary shaker (180 rpm) at 37 °C. The production of the recombinant Cel-5A was induced with 0.2 mM of isopropyl-β-thiogalactopyranoside (IPTG) at an OD_600nm of 0.6 at 37 °C for 5 h.
Bacterium culture with recombinant Cel-5A was collected by centrifugation at 10,000×g for 10 min at 4 °C and washed with 1 × PBS buffer solution (pH 7.2) twice. Cells were re-suspended in 1 × PBS buffer solution, followed by ultrasonication for 20 min using a SCIENTZ-IID ultrasonic homogenizer (Ningbo Scientz Biotechnology Polytron Technologies Inc., Zhejiang, China). The cell debris was removed by centrifugation at 15,000×g for 20 min at 4 °C. The supernatant was collected and applied to a Ni-NTA affinity chromatography column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Hybrid protein was then washed away with 40 mM imidazole and the recombinant eluted of Cel-5A protein with his-tag with 80 mM imidazole made up in 1 × PBS buffer solution (pH 7.2). The eluted protein was detected with 12% SDS-PAGE.