Selenomethionine incorporation into a protein by cell-free synthesis.
Multi-wavelength anomalous diffraction phasing is especially useful for high-throughput structure determinations. Selenomethionine substituted proteins are commonly used for this purpose. However, the cytotoxicity of selenomethionine drastically reduces the efficiency of its incorporation in in vivo expression systems. In the present study, an improved E. coli cell-free protein synthesis system was used to incorporate selenomethionine into a protein, so that highly efficient incorporation could be achieved. A milligram quantity of selenomethionine-containing Ras was obtained using the cell-free system with dialysis. The mass spectrometry analysis showed that more than 95% of the methionine residues were substituted with selenomethionine. The crystal of this protein grew under the same conditions and had the same unit cell constants as those of the native Ras protein. The three-dimensional structure of this protein, determined by multi-wavelength anomalous diffraction phasing, was almost the same as that of the Ras protein prepared by in vivo expression. Therefore, the cell-free synthesis system could become a powerful protein expression method for high-throughput structure determinations by X-ray crystallography.
Kigawa
Takanori
T
RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan.
Yamaguchi-Nunokawa
Emi
E
Kodama
Koichiro
K
Matsuda
Takayoshi
T
Yabuki
Takashi
T
Matsuda
Natsuko
N
Ishitani
Ryuichiro
R
Nureki
Osamu
O
Yokoyama
Shigeyuki
S
D002474:Cell-Free System; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D012645:Selenomethionine / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018631:ras Proteins / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism
D011506:Proteins; D012645:Selenomethionine; D018631:ras Proteins