[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]. / Poluchenie v E. coli i 19F-IaMR issledovanei 5-ftortriptofansoderzhashchego n-kontsevogo domena alpha-sub''edinitsy atsetilcholinovogo retseptora Torpedo californica.

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.