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Site-Specific Cross-Linking of In Vitro Synthesized E. coli Preproteins for Investigating Transmembrane Translocation Pathways

 Last Update: 2020-11-29
 Source: Internet
 Author: User

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proteins produced cell

coli system

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A method is described for the preparation and usage of an
E. coli
cell-free translation system primed to incorporate the commercially available photoreactive analogue of phenyalanine,
p
Bpa, into newly synthesized proteins. Incorporation is achieved by means of an amber suppressor tRNA specifically charged with
p
Bpa. The method is exemplified for the site-specific photocross-linking of the signal sequence of a Tat (twin-arginine translocation) precursor protein to the Tat translocase in the cytoplasmic membrane of
E. coli
.

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