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To-Red Photoconversion of a Fluorescent Protein

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To-Red Photoconversion of a Fluorescent Protein An optical marker based on the UV-induced green- to-red photoconversion of a fluorescent protein Ryoko Ando*†, Hiroshi Hama*, Miki Yamamoto-Hino*, Hideaki Mizuno*, and Atsushi Miyawaki*‡ *Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan; and †Brain Science Research Division, Brain Science and Life Technology Research Foundation, 1-28-12 Narimasu, Itabashi, Tokyo 175-0094, Japan Edited by Roger Y. Tsien, University of California at San Diego, La Jolla, CA, and approved August 14, 2002 (received for review May 29, 2002) We have cloned a gene encoding a fluorescent protein from a stony Materials and Methods coral, Trachyphyllia geoffroyi, which emits green, yellow, and red cDNA Cloning and Gene Construction. A BamHI–HindIII fragment light. The protein, named Kaede, includes a tripeptide, His-Tyr-Gly, encompassing the multicloning site of pFastBac1 (GIBCO͞ that acts as a green chromophore that can be converted to red. The BRL) was inserted into the BamHI͞HindIII sites of pRSETB red fluorescence is comparable in intensity to the green and is (Invitrogen), yielding pRSET-FastBac, in which an EcoRI site is stable under usual aerobic conditions. We found that the green-red located closer to the promoter than a NotI site. The stony coral conversion is highly sensitive to irradiation with UV or violet light T. geoffroyi was purchased from an aquarium shop. Whole tissue (350–400 nm), which excites the protonated form of the chro- was ground down with a MultiBeads Shocker (Yasui Kikai, mophore. The excitation lights used to elicit red and green fluo- Osaka), and total RNA was isolated by following the single- rescence do not induce photoconversion. Under a conventional step guanidinium method (8). mRNA was purified by using an epifluorescence microscope, Kaede protein expressed in HeLa cells Oligo-Tex kit (Roche Diagnostics). cDNA was synthesized turned red in a graded fashion in response to UV illumination; with an EcoRI site at the 5Ј end and a NotI site at the 3Ј end by maximal illumination resulted in a 2,000-fold increase in the ratio using a Directional Cloning Tool Kit (Amersham Pharmacia). ͞ of red-to-green signal. These color-changing properties provide a Ligation of the cDNAs into an EcoRI NotI-cleaved pRSET- simple and powerful technique for regional optical marking. A FastBac plasmid produced a directional cDNA library in a focused UV pulse creates an instantaneous plane source of red prokaryotic expression vector. The library was transformed into Kaede within the cytosol. The red spot spreads rapidly throughout the Escherichia coli strain JM109 (DE3). Colonies were screened the cytosol, indicating its free diffusibility in the compartment. The for fluorescence by using a UV illuminator (365 nm) or our extensive diffusion allows us to delineate a single neuron in a homemade fluorescence analyzing system (9). The cDNA of the coding region of Kaede (no. 9) was amplified by using dense culture, where processes originating from many different Ј Ј somata are present. Illumination of a focused UV pulse onto the primers containing 5 BamHI and 3 EcoRI sites. The restricted product was cloned in-frame into the BamHI͞EcoRI sites of soma of a Kaede-expressing neuron resulted in filling of all pro- pRSETB for bacterial expression. The 5Ј end of the Kaede gene cesses with red fluorescence, allowing visualization of contact sites was modified by PCR to have a Kozak consensus sequence between the red and green neurons of interest. (CCACCATG) after the BamHI site. The BamHI͞EcoRI frag- ment was subcloned in the mammalian expression vector he mobility of a fluorescent-labeled molecule has been pcDNA3 (Invitrogen). Tassessed by using a specific photobleaching technique called fluorescence recovery after photobleaching (1). Acquisition of a Protein Expression, in Vitro Spectroscopy, pH Titrations, SDS͞PAGE, series of images after photobleaching in a small region of the cell and Multiangle Light Scattering (MALS). Proteins were expressed in is useful for qualitative description of the diffusion of the E. coli, purified, and spectroscopically characterized as described fluorescent-labeled molecule. For studying cell lineage, or- (10). The quantum yields of green and red Kaede were deter- ganelle dynamics, and protein trafficking, however, optical mined in comparison to fluorescein (0.91). For calculation of marking is preferred; individual cells, organelles, and proteins molar extinction coefficients, protein concentrations were mea- can be optically labeled with unique colored markers. sured by using a Bradford assay kit (Bio-Rad) with BSA as the The GFP from the luminescent jellyfish Aequorea victoria standard. pH titrations were performed as described (11). ͞ (Aequorea GFP) allows direct genetic encoding of strong fluo- SDS PAGE analyses routinely used 15% polyacrylamide gels. rescence and thus is used widely in molecular and cellular MALS was measured with a multiangle light photometer biology (2). Many other GFP-like fluorescent proteins have been (DAWN, Wyatt, Santa Barbara, CA) connected to a Shodex isolated from fluorescent, but nonbioluminescent, Anthozoa HPLC system (Showa Denko, Tokyo) on which size exclusion species (3, 4). Three methods of optical marking have become columns (tandem connection of KW-804 and KW-802.5, Showa commonplace and are based on photo-induced alteration of the Denko) were installed. C-terminal His-tagged Kaede was ex- pressed in E. coli by using a pET23b vector (Novagen), and the excitation or emission spectrum of certain fluorescent proteins purified protein was analyzed. (5–7), such as WT Aequorea GFP and DsRed, a GFP-like fluorescent protein. Some limitations of these methods, how- Dissociation Culture of Hippocampal Neurons. Primary neurons were ever, have been recognized, including dimness and instability of prepared from Wistar rat embryos (embryonic day 17–18) as BIOCHEMISTRY the photoconverted product and the unavailability of simple, described (12, 13). Calcium phosphate precipitation was used to efficient, or specific illumination for marking. transfect the neurons. In this study, we have cloned a cDNA encoding a fluorescent protein from the stony coral Trachyphyllia geoffroyi. The protein emits bright green fluorescence after synthesis, but changes This paper was submitted directly (Track II) to the PNAS office. efficiently to a bright and stable red fluorescence on irradiation Abbreviations: MALS, multiangle light scattering; R͞G, red to green. with UV or violet light. In this article, we describe examples Data deposition: The sequence reported in this paper has been deposited in the GenBank demonstrating the utility of this marker, especially for cell database (accession no. AB085641). labeling. ‡To whom reprint requests should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.202320599 PNAS ͉ October 1, 2002 ͉ vol. 99 ͉ no. 20 ͉ 12651–12656 Downloaded by guest on September 30, 2021 Imaging. HeLa cells were grown on a 35-mm glass-bottom dish in Dulbecco’s modified Eagle’s medium containing 10% FBS. Cells were imaged 2–5 days after cDNA transfection with lipofectin (GIBCO͞BRL). Cells were imaged on an inverted microscope (Olympus IX70) with a standard 75-W xenon lamp and a ϫ40 objective lens (Uapo͞340, numerical aperture 1.35). Interfer- ence filters (excitation and emission filters) contained in wheels were automated by using Lambda 10-2 hardware (Sutter Instru- ments, Novato, CA). The filter-switching time was set to 50 ms. Emitted light was captured by a cooled charge-coupled device camera (Cool Snap HQ, Roper Scientific, Tucson, AZ). The whole system was controlled by using METAFLUOR 4.5 software (Universal Imaging, Media, PA). Power density of the excitation lights at the cell level was measured with a homemade power meter. Cultured neurons were observed with a confocal micro- scope based on a Fluoview FV500 scanning unit (Olympus), a diode pumped solid-state laser [Sapphire 488-20 (Coherent Radiation, Palo Alto, CA)], and a green He͞Ne laser. Green and red fluorescence signals were captured simultaneously by using the 488- and 543-nm laser lines. Results and Discussion Cloning of a Fluorescent Protein That Changes from Green to Red. Approximately 150,000 bacterial colonies containing a cDNA library prepared from T. geoffroyi were screened for fluores- cence. Two clones were selected that encoded a greenish fluo- rescent protein. Sequence analysis revealed that the two clones were identical and encoded a fluorescent protein, which was temporarily referred to as no. 9. Although the stony coral Fig. 1. Amino acid sequence (single-letter code) alignment of no. 9 (Kaede) exhibits colorful green, yellow, and red fluorescence, no colonies with avGFP (A. victoria GFP) (2), DsRed (3), and the following recently cloned emitting yellow or red light were obtained. Based on an amino fluorescent proteins (4): mcavGFP, mcavRFP, rfloGFP, and rfloRFP. The num- ␤ bering is based on Aequorea GFP. In the sequence of avGFP, ␤-sheet-forming acid sequence alignment (Fig. 1), no. 9 has a similar -can fold regions are underlined. Residues whose side chains form the interior of the to other fluorescent proteins. The crucial amino acid residues ␤-can (3) are shaded. Residues responsible for chromophore synthesis are that are involved either directly in choromophore formation indicated by *. (Tyr-66, Gly-67) or indirectly, but strongly mediate the process (Arg-96, Glu-222) are conserved in no. 9. The nearest homo- logue is mcavRFP from the great star coral Montastraea caver- peaks appeared at 572 (␧ ϭ 60,400 MϪ1⅐cmϪ1), 533, and 348 nosa (4). Both no. 9 and mcavRFP have a histidine at position nm. These peaks reflect the ionized state of the chromophore; 65; aromatic amino acids are not found in this position in any they were augmented equally with increasing pH, with a pKa fluorescent proteins except for no. 9, mcavRFP, and rfloRFP, of 5.7. When excited at 480 nm, the protein showed an emission another GFP-like protein from Ricordea florida (4).
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