Profinia affinity chromatography protein purification system

Genes encoding active TIL variants and WT indole-lyase were cloned into the NcoI/EcoRV restriction site of pETDuet-1 (Novagen) and expressed in E. coli JM109 (DE3) ∆tnaA. The recombinant cells were cultured in LB medium at 37 °C in a shaking incubator and isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM, when the optical density at 600 nm (OD₆₀₀) reached 0.4 and the cells were further incubated for 6 h. The cells were harvested by centrifugation at 5,000 rpm for 10 min. The pellet was resuspended in lysis buffer containing 50 mM sodium monophosphate, 300 mM NaCl, 10 mM imidazole and 0.1 mM phenylmethylsulfonyl fluoride as protease inhibitor, and disrupted by sonication for 3 min on ice. The lysate was centrifuged at 15,000 rpm for 10 min to remove cell debris. The supernatant was used directly to purify the His-tagged enzymes by loading into a Profinia™ Affinity Chromatography Protein Purification System (Bio-Rad, Hercules, CA, USA). The purified enzymes were confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and protein concentrations were quantified by the Bradford method. The purified enzymes were immediately examined for biochemical and kinetic characterisation.

The recombinant cells were harvested and resuspended in buffer A (50 mM sodium monophosphate, 300 mM NaCl, 10 mM imidazole, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) as a protease inhibitor). The resuspended cells were disrupted by ultrasonication (Fisher Scientific, Pittsburgh, PA) on ice. The cell debris was removed by centrifugation at 15,000 × g for 20 min at 4°C and the supernatant was filtered through a 0.45 μm filter and applied to an IMAC chromatography column (Bio-Rad, Hercules, CA) equilibrated with buffer A. The bound protein was eluted with a linear gradient between 10 mM and 250 mM imidazole in buffer A. The active fraction was dialyzed in 50 mM HEPES buffer (pH 7.5) and the resulting solution was used as the purified enzyme. All purification steps using columns were carried out using a Profinia^™ Affinity Chromatography Protein Purification System (Bio-Rad). The protein concentration was quantified by the method reported by Bradford[11 (link)]. The purified proteins were confirmed by SDS-PAGE gel analysis.

The bulk of TcpA-C (about 90%) was detected in inclusion bodies. The remaining ~10% of the protein population localized to the soluble fraction of cell lysate and was purified in two steps: (1) by affinity chromatography on Ni-NTA resin (Profinia™ Affinity Chromatography Protein Purification System; Bio-Rad Laboratories, Inc., Hercules, CA, USA), followed by (2) size-exclusion chromatography on a Superdex 200 column (GE Healthcare, Chicago, IL, USA), as described elsewhere [17 (link)]. At the first stage of purification, the soluble fraction of cell lysate was applied to the Ni-NTA column. Washing was carried out using Native IMAC Wash Buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with stepwise increasing concentrations of imidazole—30 mM (10.0 mL), 50 mM (10.0 mL), and 100 mM (10.0 mL)—to reduce nonspecific binding of contaminating proteins to the sorbent and increase the purity of the TcpA protein. Native IMAC Elution Buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with 250 mM imidazole was used in elution. At the second stage, size-exclusion chromatography on a Superdex 200 column (GE Healthcare, Chicago, IL, USA) used 20 mM Tris-HCl buffer, pH 8.9, and 150 mM NaCl. The peak containing the protein of interest with M_r of about 20 kDa was collected, concentrated up to 0.5 mg/mL, and used for the subsequent analysis.