An economical method for producing stable-isotope labeled proteins by the E. coli cell-free system.
Improvement of the cell-free protein synthesis system (CF) over the past decade have made it one of the most powerful protein production methods. The CF approach is especially useful for stable-isotope (SI) labeling of proteins for NMR analysis. However, it is less popular than expected, partly because the SI-labeled amino acids used for SI labeling by the CF are too expensive. In the present study, we developed a simple and inexpensive method for producing an SI-labeled protein using Escherichia coli cell extract-based CF. This method takes advantage of endogenous metabolic conversions to generate SI-labeled asparagine, glutamine, cysteine, and tryptophan, which are much more expensive than the other 16 kinds of SI-labeled amino acids, from inexpensive sources, such as SI-labeled algal amino acid mixture, SI-labeled indole, and sodium sulfide, during the CF reaction. As compared with the conventional method employing 20 kinds of SI-labeled amino acids, highly enriched uniform SI-labeling with similar labeling efficiency was achieved at a greatly reduced cost with the newly developed method. Therefore, our method solves the cost problem of the SI labeling of proteins using the CF.
RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan.
Jun
J
Yokoyama
Takayoshi
T
Matsuda
Seizo
S
Koshiba
Takanori
T
Kigawa
D000596:Amino Acids / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D001216:Asparagine / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
D000596:Amino Acids; D009587:Nitrogen Isotopes; D011506:Proteins; D005973:Glutamine; D001216:Asparagine; D014364:Tryptophan; D003545:Cysteine