His trap nickel chelating columns

Human ApoA-I proteins, containing a His-tag and tobacco etch virus (TEV) protease recognition site at the N terminus, were expressed in the bacterial Escherichia coli) BL21(DE3) pLysS strain (Invitrogen, Thermo Fisher Scientific) as described in the study by Del Giudice and Lagerstedt (21 (link)).
Recombinant proteins were then purified using immobilized metal affinity chromatography (His-Trap-Nickel-chelating columns, GE Healthcare) followed by treatment with TEV protease and a second immobilized metal affinity chromatography step to remove the His-tag. TEV cleavage results in 2 additional amino acid residues (Ser and Leu) at the N terminus of the purified ApoA-I proteins. Protein samples were analyzed by SDS-PAGE followed by Coomassie staining, and concentration was determined by using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). As previously shown, the purity of the recombinant lipid-free ApoA-I proteins was about 80% as determined by mass spectrometry analysis and, importantly, none of the contaminant constituted more than 1% of total protein species (21 (link)).

ApoA-I WT, Milano (R173C) and A164S proteins were produced and purified as previously described^{30 (link),31 (link)}. In short, a bacterial expression system consisting of pEXP-5 plasmid (Novagen) in Escherichia coli strain BL21(DE3) pLysS cells (Invitrogen) was used to produce the apoA-I proteins. His-tagged ApoA-I proteins were purified from bacterial cell lysate by immobilized metal affinity chromatography (His-Trap-Nickel-chelating columns, GE Healthcare) under denaturing conditions (3 M guanidine in phosphate-buffered saline (PBS), pH 7.4). Following binding, an extensive wash with 40 mM imidazole in PBS was performed and proteins were then eluted with 500 mM imidazole in PBS. Imidazole was removed from protein samples by using desalting columns (GE Healthcare) equilibrated with PBS, pH 7.4, and tobacco etch virus (TEV) protease was employed overnight at 4 °C, in the presence of 1 mM DTT, to remove the His-tag from protein samples. At the end of the incubation, a reverse Ni²⁺-column step was employed in order purify the cleaved ApoA-I from TEV protease and the His-tag. The flow-through containing cleaved ApoA-I protein was desalted into McIlvaine Buffer (165 mM Na₂HPO₄, 17.6 mM citrate, pH 7) and stored at 4 °C prior to use. SDS-PAGE and Blue-native gel (Invitrogen) were used for analyses of lipid-free protein and HDL particles.

Human ApoA-I proteins, containing at the N-terminus a 6-Histidine-tag and tobacco etch virus (TEV) protease recognition site, were produced in Escherichia coli BL21(DE3) pLysS (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) as described in [42 (link)].
The recombinant proteins were isolated using an immobilized metal affinity chromatography (IMAC, His-Trap-Nickel-chelating columns, GE Healthcare, Chicago, IL, USA) and the his-tag removed by TEV protease treatment. The his-tag free proteins were then purified by performing a second IMAC [43 (link)]. Protein purity was analyzed by SDS-PAGE, followed by Coomassie staining, and protein concentration was determined by using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific).