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Anal Biochem. 2011 Apr 15;411(2):223-9. doi: 10.1016/j.ab.2011.01.017. Epub 2011 Jan 20.

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

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  • 1RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan.

Abstract

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.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID:
21256106
[PubMed - indexed for MEDLINE]
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