Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis.
For the application of multidimensional NMR spectroscopy to larger proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism drastically reduces the efficiency and selectivity of labeling in in vivo expression systems. In the present study, a cell-free protein synthesis system was optimized, so that highly efficient and selective stable isotope labeling of proteins can be achieved in the absence of amino acid metabolism. The productivity of the E. coli cell-free coupled transcription-translation system was first improved, by about fivefold, by using the T7 RNA polymerase for transcription and also by improving the translation conditions. Thus, about 0.1 mg protein per 1 ml reaction mixture was synthesized. Then, this improved cell-free system was used for Asp- or Ser-selective 15N-labeling of the human c-Ha-Ras protein. With a 15 ml cell-free reaction, using less than 1 mg of 15N-labeled amino acid, 1 mg of the Ras protein was obtained. 1H-15N HSQC experiments confirmed that the Ras protein was efficiently labeled with high selectivity. These results indicate that this cell-free protein synthesis system is useful for NMR studies.
Kigawa
T
T
Department of Biophysics and Biochemistry, School of Science, University of Tokyo, Japan.
Muto
Y
Y
Yokoyama
S
S
D000596:Amino Acids / Q000737:chemistry*; D001224:Aspartic Acid; D002474:Cell-Free System; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D016283:Proto-Oncogene Proteins p21(ras) / Q000138:chemical synthesis / Q000737:chemistry; D012694:Serine; D014764:Viral Proteins
D000596:Amino Acids; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011506:Proteins; D014764:Viral Proteins; D001224:Aspartic Acid; D012694:Serine; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)