Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications Bruce R. Branchini a, * , Tara L. Southworth a , Neelum F. Khattak a , Elisa Michelini b , Aldo Roda b a Department of Chemistry, Connecticut College, New London, CT 06320, USA b Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, Bologna 40126, Italy Received 4 May 2005 Available online 8 August 2005 Abstract Light emission from the North American firefly Photinus pyralis, which emits yellow–green (557-nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. Here, we present studies leading to the production of a set of red- and green-emitting luciferase mutants with bioluminescent properties suitable for expanding the use of the P. pyralis system to dual-color reporter assays, biosensor measurements with internal controls, and imaging techniques. Using a combination of mutagenesis methods, we determined that the Ser284Thr mutation was sufficient to create an excellent red-emitting luciferase with a bioluminescence maximum of 615 nm, a narrow emission bandwidth, and favorable kinetic properties. Also, we developed a luciferase, containing the changes Val241Ile, Gly246Ala, and Phe250Ser, whose emission maximum was blue-shifted to 549 nm, providing a set of enzymes whose bioluminescence maxima were separated by 66 nm. Model studies demonstrated that in assays using a set of optical filters, the luciferases could be detected at the attomole level and seven orders of magnitude higher. In addition, in the presence of the Ser284Thr enzyme serving as a control, green light emission could be measured over a 10,000-fold range. The results presented here with the P. pyralis mutants provide evidence that simultaneous multiple analyte assay development is feasible with these novel proteins that require only a single substrate. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Bioluminescence; Firefly; Luciferase; Mutagenesis; Imaging; Reporter; Dual color; Red emission; Multiplex assay Luciferase from the North American firefly Photinus pyralis (Luc) 1 is a well-characterized enzyme that cata- lyzes the emission of yellow–green light [1,2]. Luc first converts the substrates firefly luciferin (LH 2 ), a hetero- cyclic carboxylic acid, and Mg-ATP into the corre- sponding luciferyl-adenylate (Eq. (1)). This reactive intermediate combines with molecular oxygen at the luciferase-active site to produce an electronically excited state product (Eq. (2)), which rapidly emits a photon of visible light (Eq. (3)): Luc þ LH 2 þ ATP ¡ Mg 2þ Luc  LH 2 -AMP þ Ppi; ð1Þ Luc  LH 2 -AMP þ O 2 ! Luc  Oxyluciferin  þ AMP þ CO 2 ; ð2Þ Luc  Oxyluciferin  ! Luc  Oxyluciferin þ ht. ð3Þ 0003-2697/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.07.015 * Corresponding author. Fax: +1 860 439 2479. E-mail address: brbra@conncoll.edu (B.R. Branchini). 1 Abbreviations used: Luc, Photinus pyralis luciferase (EC 1.13.12.7); LH 2 , D-firefly luciferin; GFP, green fluorescent protein; GST, gluta- thione-S-transferase; WT, recombinant P. pyralis luciferase containing the additional N-terminal peptide GlyProLeuGlySer–; LB, Luria– Bertani; IPTG, isopropyl-b-D-thiogalactopyranoside; CBA, 50 mM Tris–HCl (pH 7.0) containing 150 mM NaCl, 1 mM ethylenediamine tetraacetic acid, 1 mM dithiothreitol, 0.8 M ammonium sulfate, and 2% glycerol; BSA, bovine serum albumin. www.elsevier.com/locate/yabio Analytical Biochemistry 345 (2005) 140–148 ANALYTICAL BIOCHEMISTRY