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[5-fluoro-tryptophan-containing N-terminal Domain of the Alpha-Subunit of the Torpedo Californica Acetylcholine Receptor: Preparation in E. Coli and 19F NMR Study]

[5-fluoro-tryptophan-containing N-terminal Domain of the Alpha-Subunit of the Torpedo Californica Acetylcholine Receptor: Preparation in E. Coli and 19F NMR Study]

T A Alekseev, N I Dergousova, E D Shibanova, E A Azeeva, E V Kriukova, T A Balashova, P V Dubovskiĭ, A S Aesen'ev, V I Tsetlin

Bioorg Khim. Jul-Aug 2003;29(4):384-90.

PMID: 12947759

Abstract:

A protein corresponding to the extracellular 1-209 domain of the alpha-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 approximately 50%. The spectrum of the protein reduced under 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 alpha-neurotoxins was demonstrated with the use of radioiodinated alpha-bungarotoxin and trifluoroacetylated alpha-cobratoxin.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP16626021	5-Fluoro-L-tryptophan		16626-02-1	Price

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