2.3. Production of selenomethionine-modified proteins  

The pKK223.3-AtaR-AtaT-His plasmid was transformed into the methionine auxotroph E. coli strain B834(DE3) (Studier et al., 1990 ▸; Doherty et al., 1995 ▸) by electroporation. A single colony was tested for auxotrophy on minimal medium supplemented with 100 mg l⁻¹ ampicillin, 0.2% glucose with or without 50 mg l⁻¹ l-methionine. For protein expression, cells from an overnight pre-culture grown in SeMet medium (Molecular Dimensions) supplemented with 100 mg l⁻¹ ampicillin, 0.2% glucose and 50 mg l⁻¹ l-methionine were washed three times in sterile water and inoculated into fresh SeMet medium supplemented with 100 mg l⁻¹ ampicillin, 0.2% glucose and 50 mg l⁻¹ l-selenomethionine. The culture was grown at 310 K with aeration until the OD_600 nm reached 0.8, when expression of the genes was induced by the addition of 0.5 mM IPTG and continued at 301 K overnight. The cells were then harvested and the proteins were purified as described above.