Figure 7
In vitro properties of the single amino acid-substituted mutants at L304. (A) Western blotting of the recombinant mdkCSS mutants, L304N, L304Q, l304I, L304E, L304A, L304F, L304K, L304S, and WT with the anti-His antibody. The soluble forms were obtained from all the recombinant proteins expressed in E. coli. Full-length blots for A are presented in Supp_FigS14. (B) Thermostability of the L-304 mutants and WT of mdkCSS. Recombinant CSS proteins were pre-heated at 25, 35, or 45 °C for 15 min and measured for in vitro CSS activity. Relative in vitro activity of CSS was obtained as the amount of produced CMP-Neu5Ac divided by the intensity of the recombinant protein, and the value for mdkCSS without pre-heating (i.e., 25 °C) was set to 1.0. The bars represent standard deviations from three independent experiments. **indicates p < 0.005.
