Selenomethionine-Labeled Protein Production for Structure Determination

Because StQng1 lacks methionine residues, point mutagenesis was used to introduce a single methionine residue in the wild type sequence to produce selenomethionine-containing protein for de novo structure determination by seleno-MAD methods. Based on naturally occurring conservative substitutions in the bacterial Qng1 proteins (Supplementary Figure S2), residue I176 was selected for point mutagenesis to methionine. To produce selenomethionine-labelled protein, the StQng1-I176M mutant (construct MAS03G5 containing non-removable C-terminal His₆ tag) was overexpressed in E. coli C41(DE3) in M9 minimal media (Molecular Dimensions) supplemented with L-selenomethionine, and additional amino acids to suppress endogenous methionine biosynthesis (40 ). Briefly, 50 ml of overnight culture in LB media was transferred to 1-liter M9 minimal media and grown at 37°C with vigorous shaking to an optical density A₆₀₀ of 0.6. One hundred mg each threonine, lysine hydrochloride and phenylalanine, 50 mg each leucine, isoleucine and valine, and 60 mg l-selenomethionine were then added to the culture. Following 15 minutes of incubation, protein overexpression was induced by addition of IPTG to a final concentration of 0.5 mM, followed by 6 h of growth at 37°C. Cells were harvested and the protein purified as described above.