Heterologous Expression and Purification of KaPOx

The full-length KaPOx gene was synthesized with a C-terminal 6×His tag and inserted into the pET-21b(+) expression vector, in which the standard N-terminal T7-tag was excluded (BioCat). This plasmid was then transformed into chemically competent E. coli T7 Express cells (New England BioLabs) according to the standard 5-min transformation protocol. Sequencing (Microsynth) confirmed the identity of the plasmid. The cultivation of E. coli cultures was carried out routinely in terrific broth (TB) Amp⁺ buffered at pH 7.5 and supplemented with ampicillin (100 μg ml⁻¹) at 37°C. Cultures were incubated at 20°C for 20 h in the presence of 1.0% (wt/wt) lactose to induce expression of KaPOx. Cell disruption and immobilized metal affinity chromatography were carried out as previously described (61 (link)), with the adaptation of using 50 mM Tris-HCl based buffers at pH 8.0. Active fractions were pooled and dialyzed at 4°C against 50 mM potassium phosphate buffer (PPB; pH 6.5) using 7-kDa-cutoff Membra-Cel (Serva) dialysis tubing. After dialysis, the yellow KaPOx precipitate was harvested from the tube and washed twice with 50 mM PPB (pH 6.5) by centrifugation at 1,000 × g and 4°C for 120 s. Homogeneity of the purified protein was confirmed by SDS-PAGE and LC-ESI-MS peptide mapping.