Abstract
Generating sufficient quantities of labeled proteins represents a bottleneck in protein structure determination. A simple protocol for producing heavy isotope as well as selenomethionine (Se-Met)-labeled proteins was developed using T7-based Escherichia coli expression systems. The protocol is applicable for generation of single-, double-, and triple-labeled proteins (15N, 13C, and 2H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for 15N and 13C, respectively, and 75% of (non-exchangeable) hydrogen for 2H labeling. The expression yields and final cell densities (OD600 ∼16) were the same as for the production of non-labeled protein. This protocol is also applicable for Se-Met labeling, leading to Se-Met incorporation into the target protein of 70% or 90% using prototrophic or methionine auxotrophic E. coli strains, respectively.
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Acknowledgements
We are most grateful to Thorsten Lührs who pointed our attention to the great need of the “protein structure community” for efficient protein labeling protocols and also for kindly providing the expression vector pET-15b-hPrP. Special thanks are also given to Thorsten Lührs and Konrad Büssow for valuable suggestions. Partial support of the FORSYS Partner Project: “Dynamics and regulation of the metabolic balance in Escherichia coli” is greatly acknowledged.
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Li, Z., Nimtz, M. & Rinas, U. Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems. Appl Microbiol Biotechnol 92, 823–833 (2011). https://doi.org/10.1007/s00253-011-3603-x
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DOI: https://doi.org/10.1007/s00253-011-3603-x