title: Rapid in vitro screening for the location-dependent effects of unnatural amino acids on protein expression and activity.
abstract: The incorporation of unnatural amino acids (uAA) can introduce novel functional groups into proteins site-specifically, with important applications in basic sciences and protein engineering. However, uAA incorporation can impact protein expression and functional activity depending on its location within the protein-a process that is not yet completely understood and difficult to predict. Therefore, practical applications often necessitate a time-consuming optimization of uAA location by individual gene cloning, expressions, purification, and evaluations for each location tested. To address this limitation, we introduce a streamlined and versatile in vitro system to rapidly express and screen uAA-containing proteins without cumbersome cell culturing or purification procedures. We utilized this technology to simultaneously screen 24 different t4-lysozyme mutants with different uAA incorporation sites in a matter of hours, compared to weeks-long workflow of conventional methods. Screening data offered a mechanistic explanation to some effects of uAA incorporation on expression and activity. Despite these insights, rational prediction of such effects remained challenging, further confirming the value of a rapid screening approach. Biotechnol. Bioeng. 2017;114: 2412-2417. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemical Engineering, Brigham Young University, Provo, Utah.
authors.forename: Song-Min
authors.initials: SM
authors.lastname: Schinn
authors.affiliation: Department of Chemical Engineering, Brigham Young University, Provo, Utah.
authors.forename: William
authors.initials: W
authors.lastname: Bradley
authors.affiliation: Department of Chemical Engineering, Brigham Young University, Provo, Utah.
authors.forename: Ashtyn
authors.initials: A
authors.lastname: Groesbeck
authors.affiliation: Department of Microbiology and Molecular Biology, Brigham Young University, Provo, Utah.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Wu
authors.affiliation: Department of Chemical Engineering, Brigham Young University, Provo, Utah.
authors.forename: Andrew
authors.initials: A
authors.lastname: Broadbent
authors.affiliation: Department of Chemical Engineering, Brigham Young University, Provo, Utah.
authors.forename: Bradley C
authors.identifier: 0000-0003-4438-183X
authors.initials: BC
authors.lastname: Bundy
mesh_terms: D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D014176:Protein Biosynthesis / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods; D013347:Subcellular Fractions / Q000378:metabolism*
chemical_list: D000596:Amino Acids
keywords: cell free protein synthesis; in vitro screening; linear DNA; nonnatural amino acid; rapid screening; unnatural amino acid