Summary
A novel method for isotope labeling in selected amino acids is presented for use with the T7 RNA polymerase system. The protocol is illustrated with the DNA-binding domain from the E2 protein of bovine papillomavirus, BPV-1. On addition of rifampicin, protein expression occurs exclusively from the gene controlled by the T7 promoter. Since the bacteria are now dedicated to the production of E2 protein, labeling with specific amino acids is efficiently performed. For example, 10 mg/l of 15N-labeled phenylalanine is shown to be sufficient for incorporation of the label, without scrambling, and without the use of an auxotrophic strain.
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Abbreviations
- IPTG:
-
isopropylthiogalactoside
- SR media:
-
synthetic rich media
- OD600 :
-
optical density or absorbance at 600 nm
- HSQC:
-
hetero-nuclear single-quantum coherence
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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Lee, K.M., Androphy, E.J. & Baleja, J.D. A novel method for selective isotope labeling of bacterially expressed proteins. J Biomol NMR 5, 93–96 (1995). https://doi.org/10.1007/BF00227474
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DOI: https://doi.org/10.1007/BF00227474