| Autor: | V. I. Tsetlin, T. A. Balashova, E. A. Azeeva, Aesen'ev As, N. I. Dergousova, Kriukova Ev, Alekseev Ta, E. D. Shibanova, Dubovskiĭ Pv |
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| Rok vydání: | 2003 |
| Předmět: | |
| Zdroj: | Russian Journal of Bioorganic Chemistry. 29:351-357 |
| ISSN: | 1068-1620 |
| Popis: | A protein corresponding to the extracellular 1–209 domain of the α-subunit of the nicotine acetylcholine receptor from the electric organ of Torpedo californica was prepared using the corresponding cDNA domain by culturing Escherichia coli cells on a synthetic medium supplemented with 5-fluoro-L-tryptophan. The presence of a (His)6 fragment preceding the 1–209 sequence allowed purification of the protein isolated from inclusion bodies by affinity chromatography on Ni-NTA Agarose. The incorporation of 5-fluorotryptophan residues was found by 19F NMR to be ∼50%. The spectrum of the protein reduced in the denaturing conditions and subsequently reoxidized in a dilute solution under denaturing conditions in the presence of 0.05% SDS was sufficiently resolved, which allowed partial assignment of 19F resonances using the Trp60Phe mutant protein. The ability of the prepared domains to specifically bind snake α-neurotoxins was demonstrated with the use of radioiodinated α-bungarotoxin and trifluoroacetylated α-cobratoxin. |
| Databáze: | OpenAIRE |
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