Histrap ff affinity column

A pET24d vector was used for the protein construct that was overexpressed in Escherichia coli BL21 (DE3) competent cells. Cell cultures were grown in LB medium at 30°C overnight, and shaken at 180 r/min. 1% lactose monohydrate (weight/volume) was used for induction. Cells were harvested and lysed by microfluidizer (M110-L, Microfluidics), and centrifuged to pellet cell debris. The supernatant was then loaded onto a GE Healthcare HisTrapFF affinity column. The lysis and wash buffer contained 20 mM HEPES pH 8.0, 250 mM NaCl, 20 mM KCl, 20 mM MgCl2, 40 mM imidazole, while the imidazole concentration in the elution buffer was increased to 500 mM. After elution, the protein was purified by SEC using an S200 Sepharose column and GE Lifesciences AKTA Prime and Purifier systems. After purification, the proteins were concentrated using Amicon Ultra-15 spin concentrators (10 kDa cutoff point).

The PsCAT1 gene was amplified by PCR using a Pst-infected wheat cDNA sample as a template. The constructed recombinant plasmid pET15b-sumo-PsCAT1 was transformed into E. coli BL21(DE3) plysS and then protein expression was induced by 0.4 mM IPTG supplement overnight at 16°C. The collected cells were suspended in ice-cold phosphate-buffered saline (PBS) solution and lysed by sonication.
The supernatant containing the soluble proteins was analyzed by SDS-PAGE, and protein purification was performed using a HisTrap FF affinity column (GE Healthcare, Uppsala, Sweden), and identified by Western blotting analysis as described by Liu et al. (2016 (link)).
CAT activity was assayed using Catalase Assay Kit (Beyotime Biotechnology, China, Beijing) according to the manufacturer’s instructions. The optimum temperature and pH of the purified enzyme were determined after the enzyme solution samples were incubated at 20 to 70°C and pH 3 to 13 for 30 min, respectively. The effects of four metal ions (0.1 mM Mn²⁺, Zn²⁺, Cu²⁺, or Fe²⁺) on the CAT activity were measured. In addition, the dynamic curve was constructed to determine Km and Vmax values. All assays were repeated three times.

For the enzymatic characterization of TaG6PDH2, the CDS of TaG6PDH2 was introduced into the vector pET15b-sumo to generate the recombinant plasmid pET15b-sumo-TaG6PDH2, which was then introduced into Escherichia coli BL21 competent cells. Next, the positive transformants were cultured in LB medium at 37 °C to an OD₆₀₀ of 0.6, and fusion protein expression was induced using 0.1 mM IPTG overnight at 28 °C. The harvested cells were suspended in lysis buffer and lysed by sonication. The supernatant containing the soluble proteins was collected and further confirmed by a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis using Coomassie brilliant blue (CBB) staining. The purification of the TaG6PDH2 fusion protein was conducted using a His-Trap FF affinity column (GE Healthcare, Uppsala, Sweden). The enzymatic characterization of TaG6PDH2 was subsequently conducted as previously described [43 (link)]. The double-reciprocal plots were used to determine the kinetic parameters of TaG6PDH2 by testing the TaG6PDH2 activity under varying substrate concentrations. This assay was repeated at least three times.

Wild-type FB cDNA sequence (accession number NM_001710) additionally containing six histidine codons at 3′ terminus, as well as sequences for D279G, F286L, K323E, Y363A variants, and the quadruple mutant containing all aforementioned substitutions were codon optimized, synthesized and cloned into pCEP4 vector in the framework of GeneArt Gene Synthesis service by Thermo Fisher. Proteins were expressed and purified as described [23 (link)]. Briefly, vector DNA was transfected into HEK293 Freestyle cells using Freestyle Max reagent (Thermo Fisher). Conditioned FreeStyle 293 expression medium (Thermo Fisher) was collected at days 2, 4 and 7 post-transfection and stored at − 80 °C until the protein purification. The resulting proteins were purified with HisTrap FF affinity column (GE Healthcare) and elution was carried out with an imidazole gradient.

The DNA encoding the target protein with a C-terminal 6 × His affinity tag was cloned into pPIC9 vector and transformed into Pichia pastoris GS115 (Invitrogen, Carlsbad, CA). After 120 h of fermentation, the culture supernatant was collected and subjected to ammonium sulphate precipitation. The precipitate was dissolved and purified with a HisTrap FF affinity column (GE Healthcare, Uppsala, Sweden). Then the protein was deglycosylated by PGNase F and the deglycosylase was removed through HisTrap FF affinity column. The protein was further purified by anion-exchange chromatography.

Untagged full-length FC27 MSP2 was expressed and purified using a strategy specific for recombinantly expressed disordered proteins, as described previously [39 (link)]. A synthetic gene encoding 3D7 MSP2, codon optimised for expression in Escherichia coli (Genescript), was cloned into pET32a (Novagen) using KpnI and NcoI. The resulting construct contains an N-terminal thioredoxin (Trx) and His₆-tag for affinity purification. Bacterial cell pellets were lysed by heating, as for FC27 MSP2 [39 (link)]. The expressed fusion protein was isolated on a HisTrapFF affinity column (GE Healthcare), eluted with imidazole and cleaved with 1% (w/w) TEV protease. The released Trx-tag and any uncleaved fusion protein were subsequently removed by a second passage through the His-trap column. Final purification of 3D7 MSP2 was by HPLC, using a C18 column (0.9 x 25 cm, Zorbax) and a linear acetonitrile gradient in 0.1% TFA. Isotopically enriched 3D7 and FC27 MSP2 for NMR studies was prepared by growing expression cultures in M9 minimal medium, with 1 g/L ¹⁵N ammonium chloride and/or 4 g/L ¹³C glucose as the sole nitrogen and carbon sources, respectively. The final recombinant 3D7 MSP2 has an N-terminal Gly derived from the TEV cleavage site whereas recombinant FC27 MSP2 has an N-terminal Met derived from the start codon.

Wild-type C2 cDNA sequence (accession number NM_000063.5) additionally containing six histidine codons at 3′ terminus, as well as sequences for single, double, triple and quadruple variants, were codon-optimized, synthesized and cloned into pCEP4 vector in the framework of GeneArt Gene Synthesis service by Thermo Fisher (Waltham, MA, USA) as described in [16 (link)]. Plasmid DNA was transfected into HEK293 Freestyle cells (Thermo Fisher) using Freestyle Max reagent (Thermo Fisher). Conditioned FreeStyle 293 expression medium (Thermo Fisher) was collected at the 2nd, 4th and 7th days post-transfection and stored at −80 °C until the protein purification. The resulting proteins were purified with HisTrap FF affinity column (GE Healthcare) (Chicago, IL, USA), and elution was carried out with an 0.7 M imidazole gradient. Buffer was exchanged to PBS and the purity was confirmed by SDS-PAGE followed by Coomassie staining.