Figure 5: Effect of double and triple mutants on α-Gal aggregation and activity.

(a) Quantification of the enzymatic activity of WT α-Gal and double and triple mutations in transiently transfected HeLa cells. The activity of the improved mutants was ∼twofold higher than the WT protein. Statistically significant values as compared with WT are indicated: ‘***’ P< 0.001 and ‘****’ P<0.0001. Data are means from three independent experiments; error bars show s.e.m. (b) Western blot of the level of WT α-Gal and mutants in the soluble and insoluble fraction of transiently transfected HeLa cells. The fraction of the mutants that is found in the insoluble fraction is significantly lower than for WT α-Gal (barplot, result of four repeats). (c) Western blot of SEC fractions of WT and double and triple mutants in transiently transfected HeLa cells. WT α-Gal and improving mutants eluted in later fractions (12.5–14.5 ml) corresponding to the active soluble form of the protein.