Nobel Prize In Chemistry for GFP

The winners of the Nobel Prize in Chemistry for 2008 were announced this month. The award this year was given jointly to Drs. Roger Tsien, Martin Chalfie and Osamu Simomura for their discovery and development of green fluorescent proteins (GFP's). GFP's are marker genes used to monitor the activity, expression levels or position of co-expressed proteins inside living cells. The original green fluorescent protein isolated from the jellyfish Aequorea victoria with green colored emission has been improved by point mutational analysis to give other fluorescent proteins having a variety of emission wavelengths for yellow, red and even blue emission spectra. This allows for multiplexed detection schemes and even bioluminescent fluorescent energy transfer (FRET) methods. These marker genes do not require any added substrate or detection scheme for observation, and are for the most part, innocuous in the living transfected cell, although some reports of interactions of GFP labeled proteins have been reported, including interference with contractile function in muscle cells and some cytotoxic effects involving apoptotic mechanisms. It has also been reported that transgenic expression of GFP caused dilated cardiomyopathy and dose-dependent co-expression of GFP and beta-galactosidase in neurons resulted in growth retardation and premature lethality in two independent transgenic mouse lines. But these effects are atypical, and GFP and analogs are widely used in many molecular biology techniques, with over 12,000 papers published last year using it as the method of marker gene detection. For more information about GFP and it uses, please see the references below or visit our website.

• Shaner NC, Steinbach PA, Tsien RY (2005) "A guide to choosing fluorescent proteins.", Nature Methods, 2: 905-909.
• Zhang J, Campbell RE, Ting A, Tsien RY (2002) "Creating new fluorescent probes for cell biology." Nature Review Molecular Biology 3: 906-918.
• Hui-wang Ai, Nathan C. Shaner,  Zihao Cheng,  Roger Y. Tsien, and Robert E. Campbell (2007) "Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins." Biochemistry, 46: 5904-5910.
• Liu, HS, Jan, MS, Chou, CK, Chen, PH, Ke, NJ (1999) "Is green fluorescent protein toxic to the living cells?" Biochem. Biophys. Res. Commun. 260: 712-717.
• Hanazono, Y, Dunbar, C E, and Emmons, RV (1997) "Green Fluorescent Protein Retroviral Vectors: Low Titer and High Recombination Frequency Suggest a Selective Disadvantage." Hum. Gene Ther. 8(11): 1313-1319.
• Haung, WY, Aramburu, J, Douglas, PS, Izumo, S (2000) "Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy." Nat. Med. 6: 482-483.
• Krestel, H E, Mihaljevic, ALA, Hoffman, D A, and Schneider, A (2004) "Neuronal co-expression of eGFP and beta-galactosidase in mice causes neuropathology and premature death." Neurobiol. Dis. 17: 310-318.
• Agbulut O, Huet A, Niederländer N, Puceat M, Menasché P, Coirault C (2007) "Green Fluorescent Protein Impairs Actin-Myosin Interactions by Binding to the Actin-binding Site of Myosin." J. Biol. Chem., 282(14): 10465-10471.
• Agbulut, O., Coirault, C., Niederlander, N., Huet, A., Vicart, P., Hagege, A., Puceat, M., and Menasche, P. (2006) "GFP expression in muscle cells impairs actin-myosin interactions: implications for cell therapy." Nat. Meth. 3: 331.

New Red Fluorescent Luciferase Vectors

Researchers at Marker Gene have developed a new luciferase gene by utilizing point mutation and codon optimization of the luciferase sequence from the natural luciferase gene isolated from Luciola cruciata (the Japanese firefly). This novel recombinant DNA has been incorporated into vectors (M1394 and M1395) containing the highly expressing CMV and SV40 promoters for high expression activities in mammalian cells. The gene codes for an improved amino acid sequence which exhibits long-wavelength light emission (red color, EM 619 nm), as well as improved thermostability and higher expression levels in mammalian cells compared with other luciferases including the native luciferase derived from the American firefly Photinus pyralis. The expressed enzyme uses the same substrate, D-Luciferin (M0237), that is used with other luciferases, and activity can easily be detected using our convenient MarkerGene^TM Live Cell Luciferase Assay Kit (M0626). Additional substrates for use in detecting cloned activity in living tissues and in vivo include the membrane permeant D-Luciferin, ethyl ester (M0906). This makes these new vectors ideal candidates for use in vivo detection methods of cloned luciferase activity. It can also be used concurrently with vectors such as the pGL3 Photinus pyralis vectors for multiplexed detection of two cloning events in the same cell line. Transfection of the vector into eukaryotic cells may be mediated by cationic lipid compounds like Lipofectamine^TM, calcium phosphate, DEAE-dextrans, or electroporation. Similar expression vectors are under construction for use in expression of this luciferase protein in both bacterial and plant cells.  Work is also currently underway to isolate the modified luciferase protein, which will be useful in coupled enzyme assays and general enzymology methods. Marker Gene has an open business model and will accept inquiries regarding licensing or manufacturing of this new luciferase for specific biological applications. For more information about these systems, please see the references below or visit our website.

• Tatsumi H, Masuda T, Kajiyama N, Nakano E (1989) "Luciferase cDNA from Japanese firefly, Luciola cruciata: cloning, structure and expression in Escherichia coli." J. Biolumin. Chemilumin. 3(2):75-78.
• Masuda T, Tatsumi H, Nakano E (1989) "Cloning and sequence analysis of cDNA for luciferase of a Japanese firefly, Luciola cruciata." Gene. 77(2):265-270.
• de Wet JR, Wood KV, Helinski DR, DeLuca M (1985) "Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli." Proc Natl Acad Sci U S A. 82(23):7870-7873.
• Mamaev SV, Laikhter AL, Arslan T, Hecht SM, (1996) "Firefly Luciferase: Alteration of the Color of Emitted Light Resulting from Substitutions at Position 286."
• Kajiyama, N, Nakano E, (1991) "Isolation and characterization of mutants of firefly luciferase which produce different colors of light." Protein Eng. 4: 691.
• Kajiyama N, Nakano E, (1993) "Thermostabilization of Firefly Luciferase by a Single Amino Acid Substitution at
  Position 217." Biochemistry 32: 13795-1 3799.
  

NBD-piperazine as a New Lysosomal Stain

Lysosomes are intracellular organelles that are involved in a variety of cellular processes including degradation of macromolecules, recycling of membrane lipids, clearance of pathogens, drug metabolic processes and normal metabolism of proteins and lipids. Lysosomes are involved in the endocytosis, phagocytosis and autophagy pathways. As such, they have found implication in numerous medical conditions including various hereditary diseases termed lysosomal storage diseases, as well as new cancer therapy methodologies involving release of Cathepsin D during apoptosis. Because the lysosomes maintain a low pH environment, typical fluorescent staining methods using common fluorophores have been problematic, since they lose appreciable fluorescence emission below pH 7. Nitrobenzoxadiazole (NBD) is a widely used fluorophore that has the unusual environmentally sensitive property of increasing fluorescence in low pH environments. Recent work from the laboratory of Dr. Kazuhiro Ishiguro and coworkers at the Nagoya University School of Medicine have found that the common fluorescent reagent 4-nitro-7-(1-piperazinyl)-2,1,3-benzoxadiazole (NBD-piperazine, M1410) is useful in fluorescence visualization of lysosomes in live HGC-27, CW-2 and IEC-18 cell lines in culture. Comparison of staining patterns with the common LysoTracker^TM or LysoSensor^TM dyes indicated comparable staining, albeit using a higher concentration of NBD-piperazine (1 uM versus 100 nM for the MP dyes), but at a lower cost per assay. Marker Gene now carries this new lysosomal stain (Product M1410) which complements our other new metabolic lysosomal enzyme probes in the MarkerGene^TM LysoLive^TM Lysosomal Phosphatase Assay Kit (M1376) and the MarkerGene^TM LysoLive^TM Lysosomal Sulfatase Assay Kit (M1377). For more information about these systems, please see the references below or visit our website.

• Ishiguro K, Ando T, Goto H, (2008) “Novel application of 4-nitro-7-(1-piperazinyl)-2,1,3-benzoxadiazole to visualize lysosomes in live cells.”BioTechniques 45(4): 465-468.
• Ghosh PB, Whitehouse MW, (1968) “7-chloro-4-nitrobenzo-2-oxa-1,3-diazole: a new fluorigenic reagent for amino acids and other amines.” Biochem. J. 108:155-156.
• Toyo'oka TM, Ishibashi Y, Takeda K, Nakashima S, Akiyama S, Uzu S, Imai K,  (1991) “Precolumn fluorescence tagging reagent for carboxylic acids in high-performance liquid chromatography: 4-substituted-7-aminoalkylamino-2,1,3-benzoxadiazoles.” J. Chromatogr. 588:61-71.
• Toyo'oka TM, (2002) “Fluorescent tagging of physiologically important carboxylic acids, including fatty acids, for their detection in liquid chromatography.” Anal. Chim. Acta 465(1-2): 111-130.
• Santa T, Matsumura D, Huang C, Kitada C, Imai K, (2002) “Design and synthesis of a hydrophilic fluorescent derivatization reagent for carboxylic acids, 4-N-(4-N-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole (NBD-PZ-NH2), and its application to capillary electrophoresis with laser-induced fluorescence detection.”  Biomed. Chromatogr. 16:523-528.
• Fehrenbacher N, Jaattela M, (2005) “Lysosomes as targets for cancer therapy.” Cancer Res. 65:2993-2995.
• Kroemer G, Jaattela M,  (2005) “Lysosomes and autophagy in cell death control.” Nat. Rev. Cancer 5:886-897.
  

New Luciferase Inhibitors Found in Routine Marker Gene Screen

Luciferase reporter-gene assays are a commonly used technique in high-throughput screening assays for identifying target binding compounds.  But recent research from the laboratory of Professor Adriaan Ijzerman and co-workers at Leiden University in The Netherlands have found a series of new luciferase inhibitors while running an antagonistic G protein-coupled receptor luciferase reporter-gene assay.  Instead of receptor antagonists, they found the high number of apparent hits to instead be false positives caused by luciferase inhibition.  The assays were found to contain several N-pyridin-2-ylbenzamide like compounds that inhibited the luciferase activity, the best of which had an IC50 value of 1.7μM determined by an in vitro enzymatic assay. In addition, these compounds were found to be competitive inhibitors of the substrate D-luciferin. They then undertook the synthesis of several analogues to investigate the structure−activity relationships of these new luciferase inhibitors and developed an additional analog with an IC50 value of 0.069μM.  Molecular modeling studies suggested that these compounds appear to bind at the D-luciferin binding site.  A number of luciferase inhibitors are known, including the the products oxyluciferin, AMP, ATP analogues and substrate-like compounds such as D-Luciferin, 6-Methyl Ether (M0236). In addition, dissimilar compounds like pifithrin-R, lipoic acid or N-tosylphenylalanine chloromethylketone (TPCK) are also known inhibitors of luciferase. These new competitive inhibitors represent a new class of compounds for potential use in marker gene systems. For more information about these inhibitors, please see the references below, or visit our website.

• Heitman LH, van Veldhoven JPD,  Zweemer AM, Ye K, Brussee J,  Ijzerman AP, (2008) “False Positives in a Reporter Gene Assay: Identification and Synthesis of Substituted N-Pyridin-2-ylbenzamides as Competitive Inhibitors of Firefly Luciferase.” J. Med. Chem. 51(15): 4724–4729.
• Rocha S, Campbell KJ, Roche KC, Perkins ND,  (2003) “The p53-inhibitor pifithrin-alpha inhibits firefly luciferase activity in vivo and in vitro. BMC Mol. Biol., 4: 9.
• Niwa K, Ohmiya Y, (2004) “Inhibitory effect of lipoic acid on firefly luciferase bioluminescence.” Biochem. Biophys. Res. Commun.,323: 625–629.
  
• Auld DS, Southall NT,  Jadhav A,  Johnson RL, Diller, DJ,  Simeonov A, Austin CP, Inglese J, (2008) “Characterization of chemical libraries for luciferase inhibitory activity.”  J. Med. Chem. 51: 2372-2386.

• Established in 1993 at the University of Oregon Riverfront Research Park.
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