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

Noelle M Olson, Jorden A Johnson, Kerstin E. Peterson, Stephen C. Henisch, Andrew P. Marshall, Michael J. Smanski, Erin E Carlson, William C.K. Pomerantz

Research output: Contribution to journalArticlepeer-review

Abstract

Current experiments that rely on biosynthetic metabolic protein labeling with 19F 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 19F-labeled proteins as analyzed by 19F NMR and LC-MS methods. Further optimization of our system should provide a cost-effective alternative to a variety of traditional protein-labeling strategies.

Original languageEnglish (US)
Article number110014
JournalJournal of Fluorine Chemistry
Volume261-262
DOIs
StatePublished - Sep 2022

Bibliographical note

Funding Information:
LTQ LC-ion trap mass spectrometric analysis was performed in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center with support from NIH grant P30-CA077598 .

Funding Information:
We acknowledge support for this research from the NIH (R35 GM140837-02). SCH was supported by the Doctoral Dissertation Fellowship of the UMN Graduate School. DM was supported by the UMN Chemistry Department. JAJ and NMO were supported by a Biotechnology Training Grant: NIH T32GM008347. NMO was also supported by the Wayland E. Noland Excellence Fellowship of the UMN Chemistry Department.

Publisher Copyright:
© 2022 Elsevier B.V.

Keywords

  • Bromodomain
  • Fluorine NMR
  • Fluorotyrosine
  • Metabolic labeling
  • Tyrosine phenol lyase

Fingerprint

Dive into the research topics of 'Development of a single culture E. coli expression system for the enzymatic synthesis of fluorinated tyrosine and its incorporation into proteins'. Together they form a unique fingerprint.

Cite this