Inhibition of [(15)N]valine transamination during selective labeling of barstar in a T7 polymerase system.
Selective labeling of barstar by the stable (15)N isotope of the valine residue with high selectivity of the label incorporation resulting from the process of gene expression in Escherichia coli BL21(DE3) has been optimized. We have shown that alpha-aminooxyacetic acid (AOAA) significantly reduces the isotope redistribution, thus increasing the selectivity of (15)N incorporation into the synthesized protein, as detected by 2D-NMR. Quantitative measurements were used to determine the selectivity for the incorporation of isotope-labeled valine residue, which was 96% in the case using AOAA. Studies of the dynamics of barstar synthesis showed that no suppression of barstar yield is observed under the regulation of the T7 polymerase expression system by isopropylthio-beta-D-galactoside (IPTG) and rifampicin using AOAA.
Kazan Institute of Biochemistry and Biophysics, Kazan Research Center, Russian Academy of Sciences, Kazan 420111, Russia.
L V
LV
Lopukhov
A A
AA
Ponomareva
L O
LO
Yagodina
D000625:Aminooxyacetic Acid / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D012293:Rifampin / Q000494:pharmacology; D000637:Transaminases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014633:Valine / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D009587:Nitrogen Isotopes; D014764:Viral Proteins; D000625:Aminooxyacetic Acid; D007544:Isopropyl Thiogalactoside; C057438:barstar protein, Bacillus amyloliquefaciens; D000637:Transaminases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D014633:Valine; D012293:Rifampin