A77E/A79S/I81L/S168A were also constructed in order to see if
there are any additive or synergistic effects of beneficial mutation.
All mutants with N-terminal thioredoxin–hexahistidine tags were
successfully overexpressed in E. coli BL21(DE3) in the presence of
heme under IPTG induction at 16 ◦C, and purified on nickel column
as before