A novel method for selective isotope labeling of bacterially expressed proteins.
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.
Lee
K M
KM
Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02111.
Androphy
E J
EJ
Baleja
J D
JD
D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins / Q000096:biosynthesis; D012293:Rifampin / Q000494:pharmacology; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics
D004268:DNA-Binding Proteins; C051550:E2 protein, Bovine papillomavirus; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin