Fluorometric quantification of green fluorescent protein in tobacco leaf extracts Goran Robic ´ a, * , Cristiano Lacorte b , Everson A. Miranda a a Departamento de Processos Biotecnológicos, Faculdade de Engenharia Química, Universidade Estadual de Campinas, CEP 13083-970 Campinas, SP, Brazil b Laboratório de Transferência de Genes, EMBRAPA Recursos Genéticos e Biotecnologia, CEP 70770-900 Brasilia, DF, Brazil article info Article history: Received 14 January 2009 Available online 18 May 2009 Keywords: GFP Quantification Fluorescence Tobacco abstract The main use of green fluorescent protein (GFP) is as a reporter system, where the existence of the protein is usually determined visually using fluorescent microscopy. Although fluorescence-based quantification of GFP is possible, background fluorescence in plants and in plant extracts was observed by our group. Another phenomenon we observed that makes quantification difficult is the increased level of GFP fluo- rescence in Nicotiana benthamiana leaf extracts, probably the result of dimerization of GFP molecules promoted by interaction with some component(s) of tobacco extracts. In the current work, the back- ground fluorescence was minimized and the enhancement of GFP fluorescence in tobacco extracts was eliminated with the addition of urea to the measured solution so that a simple quantification assay for the GFP in the tobacco extracts could be established. Ó 2009 Elsevier Inc. All rights reserved. Proteins such as green fluorescent protein (GFP), 1 b-glucuroni- dase (GUS), and luciferase (LUC) are widely used as systems for gene expression studies and as fusion tags to monitor protein localization within the cells, commonly referred to as reporter proteins [1]. Selection of a protein for these applications should be based on its stability under different conditions (e.g., temperature, pH, salinity, denaturant concentration) as well as the reproducibility of its assay. Therefore, to correctly interpret the activity of each reporter protein, it is important to understand their intrinsic properties. Although GUS is a stable enzyme and so is a good choice for the mentioned applications, however its quantification can be affected by the pres- ence of inhibitors, such as sugars and phenolics, in the plant tissues and plant extracts [2,3]. LUC is used mostly for studying the dynam- ics of in planta gene expression because the newly formed protein is rapidly inactivated [1]. Therefore, these two proteins, GUS and LUC, are only partially suitable for recombinant protein quantification. These limitations can be largely overcome by GFP, a protein from the jellyfish Aequorea victoria that, due to its unique structure, shows a bright green fluorescence when illuminated with ultravi- olet (UV) or blue light. GFP is considered to be useful as a reporter protein or a fusion tag because it does not require either substrate or cofactors for its fluorescence, allowing the protein to be detected in vivo [4]. Another advantage is that its fluorescent properties are not hindered by most N- or C-terminal peptide or protein fusions [5,6]. Expression of GFP in plants has been optimized by different approaches, including the removal of cryptic introns that ham- pered expression of the wild-type sequence [7]. Further modifica- tions at the chromophore region led to variants with shift excitation/emission and fluorescence intensity. The currently known GFP variants can be divided into seven classes based on composition of their chromophores, with each class having a dis- tinct set of excitation and emission wavelengths ranging from 360 to 489 nm for excitation and 440 to 529 nm for emission [4,8]. These GFP versions, used as a reporter genes and fusion tags, have become very important tools in cell biology studies, including subcellular localization of proteins, protease action, transcription factor, dimerization, Ca 2+ sensitivity, cellular pH alterations, pro- tein and organelle diffusion and movement within the cell, and protein–protein interactions by fluorescence resonance energy transfer (FRET) [4,6,8–10]. In addition, synthetic GFP (sGFP, also re- ferred to as enhanced GFP [EGFP]), GFP172 and GFP157 variants [11], and wild-type GFP [5] were successfully used as a fusion tag for both monitoring and purifying recombinant proteins produced in Escherichia coli [11]. One drawback of using GFP in plant systems is the fluorescence from cell wall components, chlorophyll, and other cellular com- pounds, generally referred as endogenous fluorescence [12–14]. This endogenous fluorescence can interfere in the detection of GFP by fluorescence microscopy, particularly if the total amount of GFP molecules in a given cell is low or if these molecules are not accumulated in a particular organelle [10]. Endogenous fluo- rescence of plant extracts can also substantially interfere in their quantification [1,15]. The cell wall compounds, chlorophyll, phenolic compounds, NAD, and flavonoids are some of the com- 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.05.016 * Corresponding author. Fax: +55 19 3521 3890. E-mail address: goran@feq.unicamp.br (G. Robic ´). 1 Abbreviations used: GFP, green fluorescent protein; GUS, b-glucuronidase; LUC, luciferase; UV, ultraviolet; FRET, fluorescence resonance energy transfer; sGFP, synthetic GFP; EGFP, enhanced GFP; BSA, bovine serum albumin; sGFP(S65T), sGFP gene containing a mutation at the chromophore S65T; LB, Luria Broth; IPTG, isopropyl b-D-thiogalactoside; SDS, sodium dodecyl sulfate. Analytical Biochemistry 392 (2009) 8–11 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio