[Selective labeling of barstar in a T7 polymerase system by 15N-isotopes].
It has been shown that the use of a special growth medium enriched with amino acids and an inhibitor of aminotransferases alpha-aminooxyacetic acid makes possible the selectivity of labeling of barstar with 15N-leucine and 15N-tryptophan. The system of selective labeling, which was previously optimized with respect to the time of introducing the label relative to the time of introducing the inductor IPTG and the inhibitor of cell polymerase rifampicin, was substantially refined by the use of the transamination inhibitor. The inhibition of aminotransferases enables one to completely eliminate the redistribution of the isotope, which is a necessary step in NMR studies even if the strongly metabolizable 15N-leucine is used. The suppression of the redistribution of the isotope by alpha-aminooxyacetic acid is a successful approach to preparation of any selectively labeled proteins in the T7 polymerase system.
Lopukhov
L V
LV
Ponomareva
A A
AA
Iagodina
L O
LO
D001426:Bacterial Proteins; D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli; D007553:Isotope Labeling; D007930:Leucine; D009587:Nitrogen Isotopes; D014364:Tryptophan; D014764:Viral Proteins
D001426:Bacterial Proteins; D009587:Nitrogen Isotopes; D014764:Viral Proteins; C057438:barstar protein, Bacillus amyloliquefaciens; D014364:Tryptophan; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D007930:Leucine