Use of a Special Brazilian Red-Light Emitting Railroad Worm Luciferase In
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Perez et al. BMC Biochemistry (2017) 18:12 DOI 10.1186/s12858-017-0087-z METHODOLOGY ARTICLE Open Access Use of a special Brazilian red-light emitting railroad worm Luciferase in bioassays of NEK7 protein Kinase and Creatine Kinase Arina Marina Perez1,3†, Bruno Aquino1,3†, Vadim Viviani4 and Jörg Kobarg2,1* Abstract Background: Luciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP. Methods: Here, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP. Results: In this work we used, after several optimization reactions, creatine kinase isoforms as well as NEK7 protein kinase in the absence or presence of ATP analogous inhibitors to validate this new luminescence method. Conclusion: With this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme. Background The firefly luciferin-luciferase system is one of the best Luciferases from bioluminescent organisms have many studied ones. Beetle luciferases are bi-functional enzymes applications in biomedical areas, ranging from bioengin- with CoA-ligase and oxygenase activities [1]. The firefly eering, biosensors, cell biology to competition assays for luciferases consist of a monomer of approximately 60 kDa bioassay development. and 550 amino acids residues [3]. They catalyze the One of the most important sources of luciferases are bioluminescent reaction involving firefly luciferin (D-LH2: the bioluminescent beetles belonging to the Elateroidea a benzothiazolic compound), adenosine triphosphate superfamily, within Lampyridae (fireflies), Phengodidae (ATP), magnesium ion and molecular oxygen with the for- (railroadworms) and Elateridae (click-beetles) [1, 2]. mation of an electronically excited species (oxyluciferin), They emit different bioluminescence colors depending inorganic pyrophosphate (PPi), carbon dioxide and adeno- on life stage and the lantern type, from green to red, that sine monophosphate (AMP) [1]. serve as ecological adaptations to different photic Firefly luciferase catalyzed reaction: environments and for diverse biological functions. Luciferase þ ATP þ luciferin þ O2→AMP þ PPi þ Oxyluciferin þ CO þ light * Correspondence: [email protected] 2 Vadim Viviani and Jörg Kobarg: these two authors co-supervised the project. † Equal contributors This reaction follows the Michaelis– Menten equation 2Faculdade de Ciências Farmacêuticas, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil for both ATP and luciferin substrates and can be used to 1Instituto de Biologia, Departamento Bioquímica e Biologia Tecidual, quantitatively measure the ATP reaction. Universidade Estadual de Campinas, Campinas, Programa de Pós-gradução The firefly luciferin-luciferase reaction became widely em Biologia Molecular e Funcional São Paulo, Rua Monteiro Lobato 255, Campinas, SP CEP 13083-862, Brazil used for analytical purposes because light can be easily Full list of author information is available at the end of the article detected with high sensitivity and with simple © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Perez et al. BMC Biochemistry (2017) 18:12 Page 2 of 8 instrumentation. Furthermore, light emission can be isopropyl-β-D-thio-galactoside (IPTG), and purified measured in different types of vessels, even inside cells, according established procedures. After expression, the living animals or plants, because ATP provides the acti- 6xHis-red luciferase fusion protein was purified using a vation energy for all living cells and is used as cofactor Hitrap chelating HP column. The column and the lysate by hundreds of enzymes. were equilibrated in Buffer A (20 mM sodium phosphate Throughout evolution, cells have adapted the KM values pH 8.0; 0.5 M NaCl; 20 mM imidazole) and eluted in of most of their enzymes to allow efficient regulation of Buffer B (20 mM sodium phosphate pH 8.0, 0.5 M NaCl, their enzyme activities. Consequently, most living cells 500 mM imidazole). The eluate containing red luciferase have similar intracellular ATP concentrations. The quan- was dialyzed to Tris-HCl buffer (pH 8.0). After purifica- tity of this molecule per cell is therefore mainly tion the luciferase preparation was tested for the absence determined by the intracellular volume: normal bacterial of kinase or other ATP consuming enzyme activities, cells contain 1–2 attomoles ATP, whereas larger mamma- which may have been co-purified from E. coli lysate lian cells typically contain 10,000–100,000 attomoles [4]. (Additional file 1: Figure S1). Due to their high bioluminescence efficiency (15– 61%), beetle luciferases, especially firefly luciferases and Creatine Kinase expression their genes, have been extensively used in analytical, Genes coding for 6xHis-brain (CKB) and 6xHis-muscle pharmaceutical, biomedical, and diagnostic applications, (CKM) creatine kinase were subcloned from pEF1α [11] and as reporter genes in molecular biology. High- with EcoRI/NotI into plasmid pET28a. Plasmids contain- throughput screening, determination of ATP, microbial ing 6xHis-sarcomeric (pUS01) and 6xHis-ubiquitous detection, immunoassays, nucleic acid assays, nucleotide (pUS04) cloned creatine kinase in pET3b vector were analysis, biosensing and bioimaging are some of the kindly provided by Dr. Schlattner [12]. All four plasmids common applications of luciferase [5–10]. However, were transformed in E. coli BL21(DE3) (Novagen) and most assays use firefly luciferases that emit in the protein expression was initiated by adding 0.5 mM of yellow-green region and are pH-sensitive, thereby in IPTG followed by incubation for 16 h at 30 °C. Cells some cases limiting the applicability to samples. Thus were harvested and ressuspended in Buffer CK-A the cloning of South-American Phrixotrix rail roadworm (50 mM Tris-HCl, pH 7.4; 250 mM NaCl; 20 mM red light luciferase increased the range of applications, Imidazole; 50 μg/ml lysozyme) for 1 h, prior sonication. extending it to usage in cultured mammalian cells, Lysates were centrifuged at 20.000 x g for 40 min at 4 °C which are regularly grown in culture medium that and proteins were purified using a HiTrap Chelating HP contains phenol red, a compound that absorbs light in column. Elution was performed using a gradient of the yellow/green parts of the spectrum. imidazole. Purified proteins were dialyzed against Buffer Among beetle luciferases, the red-emitting luciferase CK-A without imidazole and frozen at −80 °C until of Phrixotrix hirtus railroad worm, previously cloned usage. and characterized by Viviani et al. [6] is the only lucifer- ase which naturally emits red bioluminescence, with the NEK7, Mat1, NEK9 and CC2D1A expression most red-shifted and narrowest emission spectrum The GST-tagged protein fragments/domains, as re- (623 nm). Furthermore, this luciferase displays one of trieved from yeast-two hybrid screen [13]:NEK9(764– the lowest KM values for luciferin [7, 8, 10]. These prop- 976), CDK-activating kinase assembly factor MAT1(Full- erties make this enzyme especially suitable for ATP as- length) and CC2D1A(501–940) were expressed in E. coli says in pigmented samples. The aim of this paper was to BL21 (DE3) cells using 1 mM IPTG, at 25 or 30 °C, re- develop a simple, low cost, “all in-one-reaction tube”, spectively, for 4 h. The full-length human NEK7 protein competition assay to evaluate substrates and inhibitors (6 × His-NEK7) was expressed in E. coli BL21 (DE3) for a broad spectrum of kinases, represented here by the cells, as described previously [13]. Expression was NEK7 protein kinase and the creatine kinase, using P. induced for 4 h using 1 mM IPTG at 28 °C. Induced hirtus red light emitting luciferase. cells were harvested and lysed by sonication in extrac- tion buffer (50 mM HEPES, pH 7.5; 5 mM sodium Methods phosphate, 300 mM NaCl, 5% glycerol, 1 mM PMSF, Cloning and recombinant protein expression 625 μg/mL lysozyme). The cell lysates were clarified by Red emitting luciferase expression centrifugation with 16.000×g for 10 min at 4 °C in order The gene that encodes red light emitting luciferase was to obtain the supernatant. Cleared fractions were puri- originally cloned from P. hirtus railroad worm luciferase fied by GST affinity chromatography using a Glutathione [6], sub-cloned in the pCold vector [3, 6] and provided Sepharose 4 Fast Flow resin. GST fusion proteins were by one of the authors (V.V.). The luciferase was eluted in buffer containing 50 mM Tris-HCl and 50 mM expressed in E. coli BL21 cells in presence of 0.3 mM of reduced glutathione at pH 8.0. Cleared lysates Perez et al. BMC Biochemistry (2017) 18:12 Page 3 of 8 containing 6 × His-NEK7, were purified by affinity liquid HCl, pH 8; 4 mM MgSO4) in absence or presence of chromatography, using a HiTrap Chelating Affinity 0.5 μM protein substrate (NEK9). The inhibitors were Chromatography Column (GE Healthcare), followed by added in DMSO. In controls (columns 1–3), we added elution with a linear concentration gradient of imidazole the same final concentration of DMSO (0.8%).