Hiprep 26 60 sephacryl s 200 hr

The cDNA of wild-type human GCAP1-E6S (Uniprot: P43080) was cloned in a pET-11a plasmid between NdeI and NheI restriction sites (Genscript). The E6S variant was introduced to allow post-translational myristoylation on the N-terminal Gly residue by Saccharomyces cerevisiae N-myristoyl transferase (yNMT) [67 (link)]. The N104K-G105R variants were introduced by site-directed mutagenesis on a pET-11a-GCAP1-E6S plasmid (Genscript).
GCAP1 variants were heterologously expressed in Escherichia coli BL21-DE3 cells previously co-transformed with pBB131-yNMT and purified after a sequence of size exclusion chromatography (SEC, HiPrep 26/60 Sephacryl S-200 HR, GE Healthcare) and anionic exchange chromatography (AEC, HiPrep Q HP 16/10, GE Healthcare) as previously described [12 (link),67 (link)], with the only modification consisting of pH = 8 in AEC buffers. Quantification of proteins was achieved by Bradford assay [68 (link)], using a GCAP1-specific reference curve based on amino acid hydrolysis analysis (Alphalyze). Protein purity was checked on a 15% SDS-PAGE gel, GCAP1 samples were either exchanged against 20 mM Tris-HCl pH 7.5, 150 mM KCl, 1 mM DTT buffer and frozen with liquid nitrogen, or against decalcified 50 mM NH₄HCO₃ and lyophilized. Samples were finally stored at −80 °C until use.

The gene encoding CDSecA2 (UniProt: Q183M9; KEGG ID: CD630 27920) was cloned into the pMCSG7 ligation-independent vector [60 (link)] and overexpressed in E. coli BL21 (DE3) cells. Cultures were grown in 2 L of ZYM-5052 autoinduction medium with 50 mg/mL ampicillin as described by [61 (link)]. Cells were harvested and lysed, after which the lysate was separated by centrifugation and purified by HisTrap FF and HiPrep 26/60 Sephacryl S-200HR (GE Healthcare Life Sciences, Chicago, IL, USA) columns. Fractions of the resulting recombinant protein, CDSecA2 (781 residues, 89.1 kDa) containing the native sequence with the N-terminal hexa-histidine tag, and the additional sequence for protease cleavage were combined, and a recombinant His6-tobacco etch virus protease (1:50 (w/w)) was used to remove the His tag over the course of a 2-day incubation at 4 °C with 10 mM β-mercaptoethanol. After a secondary IMAC step using Ni Sepharose 6 Fast Flow (GE Healthcare Life Sciences, Chicago, IL, USA), the flow-through fraction containing the protein of interest was collected and concentrated in 30 mM Tris, 400 mM NaCl (pH 7.5), 10 mM imidazole and 10 mM β-mercaptoethanol to 2.3 mg/mL. For cocrystallization with the ATP-γ-S, β-mercaptoethanol was omitted.