Figure 2

Production, thermal stability, and activity of wild-type (WT), polymorphisms, and clinical mutations of CAVA. WT⁺ and WT⁻ indicate the presence and absence, respectively, of the N-terminal His₆ tag in the WT enzyme. In all polymorphisms and mutant forms, the N-terminal His₆ tag has been removed by tobacco etch virus (TEV) treatment. Polymorphisms are labeled in gray and underlined. (a) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (15% polyacrylamide, Coomassie blue staining) of purified human recombinant CAVA, either WT or carrying the indicated mutations. St, protein markers (PageRuler Plus prestained protein; Thermo Scientific), with masses indicated in kilodaltons. The black arrowhead indicates the position of the tag-free CAVA band of the WT form, polymorphisms, and missense mutations. The pink arrowhead indicates the position of the tag-free CAVA band of the enzyme forms carrying deletions of exon 4 or 6, respectively. (b and c) Thermostability of CAVA forms. (b) The change in T_m values (ΔT_m, compared with WT⁻) for each CAVA mutant. Error bars depict SEM from at least six measurements. *Statistically different (P < 0.05, Mann–Whitney test). (c) Differential scanning fluorimetry melting curves of WT CAVA and the three most unstable clinical mutations. (d) Enzyme activity per milligram of pure CAVA, expressed relative to the corresponding activity of the pure WT enzyme (438.4 U/mg). Error bars depict the SEM from at least six measurements. *Statistically different (P < 0.05, Mann–Whitney test).