Figure 5. Expression and analysis of mutant derivatives of His₆–BjHMGS1.

(A) Coomassie Blue-stanied SDS/PAGE of Ni²⁺-NTA column-purified mutant proteins: S89A (lane 1), K90A (lane 2), N115A (lane 3), R157A (lane 4) H188N (lane 5), C212S (lane 6), S356A (lane 7) and S359A (lane 8). Lane M, protein standards including phosphorylase (97 kDa), BSA (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (30 kDa). Five micrograms of purified proteins were loaded per well. (B) Specific activities of mutant His₆–BjHMGS1 enzymes. Enzyme activities were measured in the presence of 250 ng of purified mutant proteins in 50 mM Tris/HCl (pH 7.5) and equimolar concentrations of [¹⁴C]acetyl-CoA and AcAc-CoA at 30 °C for 10 min. The enzyme activity of mutants is expressed relative to the activity of wild-type His₆–BjHMGS1.