The Development of Biosynthesis of 2Н- and 13С-labeled Amino acids and Proteins with Various Levels of Isotopic Enrichment Using Bacterial Objects
Authors: Oleg Mosin, Ignat Ignatov, Dmitry Skladnev, Vitaly Shvets
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By the method of microbiological synthesis were obtained and analyzed by electron impact mass-spectrometry 2H, 13C-labeled amino acids of a facultative methylotrophic bacterium Brevibacterium methylicum and an obligate methylotrophic bacterium Methylobacillus flagellatum and 2H, 13C-labeled amino acids of the total protein of biomass obtained on media containing as a source of stable isotopes [2H]methanol, [13C]methanol and 2H2O. It was also performed the incorporation of L-[2,3,4,5,6-2Н]phenylalanine, L-[3,5-2Н]tyrosine and L-[2,4,5,6,7-2Н]tryptopan into the membrane integral protein bacteriorhodopsin synthesised in purple membranes of photo-organotrophic halobacterium Halobacterium halobium ET 1001. For mass-spectrometric analysis the multicomponential mixtures of 2H- and 13C-labeled amino acids, derived from cultural media and protein hydrolysates after hydrolysis in 6 M 2HСl (3% phenol) and 2 M Ва(OH)2 were modified into N-benzyloxycarbonyl-derivatives of amino acids as well as into methyl esters of N-5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl) derivatives of [2H, 13С]amino acids, which were preparative separated using a method of reverse-phase HCLP. Biosynthetically obtained 2H- and 13C-labeled amino acids represented the mixtures differing in quantities of isotopes incorporated into molecule. The levels of 2H and 13С enrichment of secreted amino acids and amino acid resigues of protein were found to vary from 20,0 atom.% to L-leucine/isoleucine up to 97,5 atom.% for L-alanine depending on concentration of 2H- and 13C-labelled substrates.