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 examples, purified tryparedoxin 1 from Crithidia fasciculata, alkylhydroperoxide 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 analysis reveals no differences between native and SeMet-labelled tryparedoxin 1 enzyme. Both proteins yield crystals under similar conditions. The culture conditions and host vector systems described greatly facilitate selenium-labelling of proteins for 3-D structure determination.
Guerrero
S A
SA
Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Germany.
Hecht
H J
HJ
Hofmann
B
B
Biebl
H
H
Singh
M
M
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D016831:Crithidia fasciculata / Q000378:metabolism; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D019910:Mycobacterium marinum / Q000378:metabolism; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism; D054464:Peroxiredoxins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012645:Selenomethionine / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism
D000942:Antigens, Bacterial; D003470:Culture Media; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C114110:tryparedoxin; C072681:Mycobacterium tuberculosis antigens; D013879:Thioredoxins; D012645:Selenomethionine; D010544:Peroxidases; D054464:Peroxiredoxins; C515937:ahpC protein, E coli