title: Multiply mutated Gaussia luciferases provide prolonged and intense bioluminescence.
abstract: Gaussia luciferase (GLuc) from the copepod Gaussia princeps is both the smallest and brightest known luciferase. GLuc catalyzes the oxidation of coelenterazine to produce an intense blue light but with a very short emission half-life. We report mutated GLucs with much longer luminescence half-lives that retain the same initial intensity as the wild-type enzyme. The GLuc variants were produced using cell-free protein synthesis to provide high yields and rapid production of fully active product as well as simple non-natural amino acid substitution. By incorporating homopropargylglycine and attaching PEG using azide-alkyne click reactions, we also show that the four methionines in GLuc are surface accessible. The mutants provide a significantly improved reporter protein for both in vivo and in vitro studies, and the successful non-natural amino acid incorporation and PEG attachment indicate the feasibility of producing useful bioconjugates using click attachment reactions.
authors.affiliation: Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stanford, CA 94305-5025, USA. jpwelsh@stanford.edu
authors.forename: John P
authors.initials: JP
authors.lastname: Welsh
authors.forename: Kedar G
authors.initials: KG
authors.lastname: Patel
authors.forename: Karthish
authors.initials: K
authors.lastname: Manthiram
authors.forename: James R
authors.initials: JR
authors.lastname: Swartz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D033342:Copepoda / Q000201:enzymology*; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D049449:Luminescence; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009154:Mutation
chemical_list: D050505:Mutant Proteins; D008156:Luciferases