A practical method for cell-free protein synthesis to avoid stable isotope scrambling and dilution.
During recent years, the targets of protein structure analysis using nuclear magnetic resonance spectroscopy have become larger and more complicated. As a result, a complete and precise stable isotope labeling technique has been desired. A cell-free protein synthesis system is appropriate for this purpose. In the current study, we achieved precise and complete (15)N and (2)H labeling using an Escherichia coli cell extract-based cell-free protein synthesis system by controlling the metabolic reactions in the system with their chemical inhibitors. The addition of aminooxyacetate, d-malate, l-methionine sulfoximine, S-methyl-l-cysteine sulfoximine, 6-diazo-5-oxo-l-norleucine, and 5-diazo-4-oxo-l-norvaline was quite effective for precise amino acid-selective (15)N labeling even for aspartic acid, asparagine, glutamic acid, and glutamine, which generally suffer from severe isotope scrambling and dilution when using the conventional cell-free system. For (2)H labeling, the back-protonation of the H(α) and H(β) positions, which commonly occurred in the conventional system, was dramatically suppressed by simply adding aminooxyacetate and d-malate to the cell-free system except for the H(α) positions in methionine and cysteine.
RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan.
Jun
J
Yokoyama
Takayoshi
T
Matsuda
Seizo
S
Koshiba
Naoya
N
Tochio
Takanori
T
Kigawa
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D002474:Cell-Free System; D003903:Deuterium / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000596:Amino Acids; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D003903:Deuterium