PMID:21983707
| Citation |
Li, Z, Nimtz, M and Rinas, U (2011) Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems. Appl. Microbiol. Biotechnol. 92:823-33 |
|---|---|
| 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 ((15)N, (13)C, and (2)H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for (15)N and (13)C, respectively, and 75% of (non-exchangeable) hydrogen for (2)H 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. |
| Links |
PubMed Online version:10.1007/s00253-011-3603-x |
| Keywords |
Carbon Isotopes/metabolism; Deuterium/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Gene Expression; Genetic Vectors; Isotope Labeling/methods; Nitrogen Isotopes/metabolism; Proteins/chemistry; Proteins/genetics; Proteins/metabolism; Selenomethionine/metabolism |
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