Folding and activity of mutant cystathionine β-synthase depends on the position and nature of the purification tag: Characterization of the R266K CBS mutant

► Nature and position of affinity tag is critical for mutant CBS folding and assembly. ► C-terminal 6xHis-tagged WT and R266K CBS has the highest activity among all constructs. ► The heme in R266K mutant is more susceptible to ligand switch than WT. ► The R266K mutation most likely destabilizes the thiolate ligand of the heme.