Two-strain, cell-selective protein labeling in mixed bacterial cultures.
Two-strain, cell-selective protein labeling in mixed bacterial cultures.
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DOI：
10.1021/ja3004667
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发表时间：
2012-05-23
影响因子：
15
通讯作者：
Tirrell, David A.
中科院分区：
文献类型：
--
作者：
Truong, Frank;Yoo, Tae Hyeon;Lampo, Thomas J.;Tirrell, David A.
Cell-selective metabolic labeling of proteins with non-canonical amino acids enables the study of proteomic changes in specified subpopulations of complex multi-cellular systems. For example, azidonorleucine (Anl) and 2-aminooctynoic acid, both of which are activated by an engineered methionyl-tRNA synthetase (designated NLL-MetRS), are excluded from proteins made in wild-type cells but incorporated readily into proteins made in cells that carry NLL-MetRS. To expand the set of tools available for cell-selective metabolic labeling, we sought a MetRS variant capable of activating propargylglycine (Pra). Pra was chosen as the target amino acid because its alkynyl side chain can be selectively and efficiently conjugated to azide-functionalized fluorescence probes and affinity tags. Directed evolution, using active-site randomization and error-prone PCR, yielded a MetRS variant (designated PraRS) capable of incorporating Pra at near-quantitative levels into proteins made in a Met-auxotrophic strain of Escherichia coli cultured in Met-depleted media. Proteins made in E. coli strains expressing PraRS were labeled with Pra in Met-supplemented media as shown by in-gel fluorescence after conjugation to Cy5-azide. The combined use of NLL-MetRS and PraRS enabled differential, cell-selective labeling of marker proteins derived from two bacterial strains co-cultured in media supplemented with Met, Anl, and Pra. Treatment of the mixed marker proteins by sequential strain-promoted and copper (I)-catalyzed cycloadditions allowed straightforward identification of the cellular origin of each protein.
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影响因子：
18.3
作者：
Ngo, John T.;Tirrell, David A.
通讯作者：
Tirrell, David A.
影响因子：
14.8
作者：
Link, A. James;Vink, Mandy K. S.;Tirrell, David A.
通讯作者：
Tirrell, David A.
影响因子：
3.5
作者：
Kiick, KL;Weberskirch, R;Tirrell, DA
通讯作者：
Tirrell, DA
影响因子：
5.6
作者：
SCHMITT, E;MEINNEL, T;MECHULAM, Y
通讯作者：
MECHULAM, Y
影响因子：
14.8
作者：
Ngo, John T.;Champion, Julie A.;Tirrell, David A.
通讯作者：
Tirrell, David A.