Development of a single culture E. coli expression system for the enzymatic synthesis of fluorinated tyrosine and its incorporation into proteins.

Autor: Olson NM; Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis MN 55455, USA., Johnson JA; Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis MN 55455, USA., Peterson KE; Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis MN 55455, USA., Heinsch SC; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108., Marshall AP; Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis MN 55455, USA., Smanski MJ; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108., Carlson EE; Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis MN 55455, USA., Pomerantz WCK; Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis MN 55455, USA.
Jazyk: angličtina
Zdroj: Journal of fluorine chemistry [J Fluor Chem] 2022 Sep; Vol. 261-262. Date of Electronic Publication: 2022 Jun 28.
DOI: 10.1016/j.jfluchem.2022.110014
Abstrakt: Current experiments that rely on biosynthetic metabolic protein labeling with 19 F often require fluorinated amino acids, which in the case of 2- and 3-fluorotyrosine can be expensive. However, using these amino acids has provided valuable insight into protein dynamics, structure, and function. Here, we develop a new in-cell method for fluorinated tyrosine generation from readily available substituted phenols and subsequent metabolic labeling of proteins in a single bacterial expression culture. This approach uses a dual-gene plasmid encoding for a model protein BRD4(D1) and a tyrosine phenol lyase from Citrobacter freundii , which catalyzes the formation of tyrosine from phenol, pyruvate, and ammonium. Our system demonstrated both enzymatic fluorotyrosine production and expression of 19 F-labeled proteins as analyzed by 19 F NMR and LC-MS methods. Further optimization of our system should provide a cost-effective alternative to a variety of traditional protein-labeling strategies.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Databáze: MEDLINE
Externí odkaz: