Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using <Emphasis Type="Italic">Escherichia coli</Emphasis>-based expression systems |
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Authors: | Zhaopeng Li Manfred Nimtz Ursula Rinas |
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Institution: | (1) Helmholtz Centre for Infection Research (SB), Braunschweig, Germany;(2) Technical Chemistry - Life Science, Leibniz University of Hannover, Hannover, Germany; |
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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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