Production of selenomethionine-labelled proteins using simplified culture conditions and generally applicable host/vector systems

The amino acid analogue selenomethionine (SeMet) is shown to be efficiently incorporated into recombinant proteins expressed in Escherichia coli grown in a simple minimal medium without the addition of synthetic amino acids. Furthermore, satisfactory SeMet incorporation is obtained with a methionine -prototrophic strain transformed with commonly used vector systems. As exam ples, purified tryparedoxin 1 from Crithidia fasciculata, alkyllrydroperoxi de reductase (AhpC) from Mycobacterium marinum and the 16-kDa antigen from M. tuberculosis are shown to be efficiently labelled with SeMet, using the culture conditions and the host/vector systems described here. Enzymatic an alysis reveals no differences between native and SeMet-labelled tryparedoxi n 1 enzyme. Both proteins yield crystals under similar conditions. The cult ure conditions and host vector systems described greatly facilitate seleniu m-labelling of proteins for 3-D structure determination.