title: An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.
abstract: Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies.
authors.affiliation: Department of Molecular Genetics and Microbiology, The University of Texas at Austin, 1 University Station, A4800, Austin, TX 78712, United States; Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station, A4800 Austin, TX 78712, United States.
authors.forename: Amrita
authors.initials: A
authors.lastname: Singh-Blom
authors.affiliation: Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station, A4800 Austin, TX 78712, United States; Applied Research Laboratories, The University of Texas at Austin, 10000 Burnet Rd. Austin, TX 78758, United States. Electronic address: randall.a.hughes@gmail.com.
authors.forename: Randall A
authors.initials: RA
authors.lastname: Hughes
authors.affiliation: Department of Molecular Genetics and Microbiology, The University of Texas at Austin, 1 University Station, A4800, Austin, TX 78712, United States; Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station, A4800 Austin, TX 78712, United States; Applied Research Laboratories, The University of Texas at Austin, 10000 Burnet Rd. Austin, TX 78758, United States. Electronic address: andy.ellington@mail.utexas.edu.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ellington
mesh_terms: D001710:Biotin; D002474:Cell-Free System / Q000502:physiology*; D004926:Escherichia coli; D008958:Models, Molecular; D014176:Protein Biosynthesis / Q000502:physiology*; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D019809:Streptavidin; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D011506:Proteins; C026927:5-fluorotryptophan; C001244:6-fluorotryptophan; D001710:Biotin; D014364:Tryptophan; D019809:Streptavidin
keywords: Cell-free protein synthesis; Residue-specific incorporation; Streptavidin; Tryptophan; Unnatural amino acids