Biosynthesis of Deuterium-labeled Transmembrane Protein Bacteriorhodopsin from Halobacterium halobium

Abstract

The semi-preparative biosynthesis of photochrome transmembrane protein bacteriorhodopsin (output 8-10 mg), labeled with deuterium on functionally important amino acid residues - [2,3,4,5,6-²H₅]phenylalanine, [3,5-²H₂]tyrosine, and [2,4,5,6,7-²H₅]tryptophan was carried out with using a photo-organotrophic halobacterium Halobacterium halobium. The protein was isolated from purple membranes by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, with the subsequent solubilization of final product with 0.5% (w/v) SDS-Na and fractionation by methanol. The homogeneity of the synthesized product, and the selectivity of deuterium incorporation into the molecule was proved by combination of preparative and analytical protein methods including elecrtophoresis in 12.5% (w/v) PAAG with 0.1% (w/v) SDS-Na, gel filtration chromatography on Sephadex G-200, and electron impact mass-spectrometry of methyl esters of N-Dns-[²H]derivatives of amino acids after their separation by reverse-phase HPLC.

Keywords: Halobacterium halobium, bacteriorhodopsin, [2,3,4,5,6-²H₅]Phe, [3,5-²H₂]Tyr, [2,4,5,6,7-²H₅]Trp, biosynthesis, EI mass-spectrometry, RP-HPLC.