A practical method for cell-free protein synthesis to avoid stable isotope scrambling and dilution

作者：Yokoyama Jun; Matsuda Takayoshi; Koshiba Seizo; Tochio Naoya; Kigawa Takanori^*

来源：Analytical Biochemistry, 2011, 411(2): 223-229.

DOI：10.1016/j.ab.2011.01.017

• Cell-free protein synthesis
• NMR
• Stable isotope labeling
• Amino acid-selective labeling
• Deuterium labeling

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 N-15 and H-2 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 N-15 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 H-2 labeling, the back-protonation of the H-alpha and H-beta 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-alpha positions in methionine and cysteine.

• 出版日期2011-4-15