Marker Gene Monthly Newsletter   

April, 2007

Volume 7, Number 4

© Copyright MGT, Inc., 2007.  Published by Marker Gene Technologies, Inc., The University of Oregon Riverfront Research Park, 1850 Millrace Drive, Eugene, Oregon 97403-1992 USA.  All rights reserved.  For information on the use or copying of the material contained in this document, please contact us at techservice@markergene.com.  Please see below for subscription information and updates.  This newsletter is labeled as an ADVERTISEMENT in accordance with the CAN-SPAM act of 2003, S.877 Public Law: 108-187.

Anaerobic GFP Proteins.

Fluorescent marker genes such as green fluorescent protein (GFP) from the jellyfish Aequorea victoria or their variants YFP and RFP have become very useful for imaging studies of protein binding, intracellular trafficking and localization, or gene expression levels in a variety of live cell assays. However, their use is generally restricted to aerobic systems, as the formation of their chromophores in vivo requires oxygen. Recently, work from the laboratory of Dr. Karl-Erich Jaeger at the Institute of Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorfand and co-workers have developed several new blue and yellow fluorescent proteins that can function in anaerobic conditions.

Starting with the blue-light photoreceptor YtvA from the bacterium Bacillus subtilis, utilizing site-directed mutagenesis to increase the fluorescence emission and incorporating the SB2 protein from the bacterium Pseudomonas putida that contains a light oxygen-voltage sensing domain, they engineered several flavin mononucleotide–based fluorescent protein systems (termed FbFPs) that can be used as fluorescent marker genes in the absence of oxygen. These new proteins can have several useful applications including high-throughput screening for anaerobic bacteria, new biosensor technologies and the development of cancer therapies involving the use of anaerobic bacteria as anti-tumor agents. For more information about these exciting new fluorescent proteins, please see the references below, or visit our website.

• Thomas Drepper, Thorsten Eggert, Franco Circolone, Achim Heck, Ulrich Krau, Jan-Karl Guterl, Marion Wendorff, Aba Losi, Wolfgang Gärtner, Karl-Erich Jaeger (2007) "Reporter proteins for in vivo fluorescence without oxygen" Nature Biotechnology 25: 443 - 445.
• Buttani, V., Losi, A., Eggert, T., Krauss, U., Jaeger, K.-E., Cao, Z., and Gaertner, W. (2007) "Conformational analysis of the blue-light sensing protein YtvA reveals a competitive interface for LOV-LOV dimerization and interdomain interactions." Photochem. Photobiol. Sci. 6:41-49.
• Krauss, U., Losi, Gärtner, W., Jaeger, K.-E., Eggert, T. (2005) "Initial characterization of a blue-light sensing phototropin-related protein from Pseudomonas putida: a paradigm for an extended LOV construct." Phys. Chem. Chem. Phys.7: 2804-2811.
• Losi, A., Polverini, E., Quest, B. & Gärtner, W. "First evidence for phototropin-related blue-light receptors in prokaryotes." Biophys. J. 82, 2627-2634 (2002).

Gaussia Luciferase DNA Assays.

Gaussia Luciferase (GLuc) is a new chemiluminescent reporter encoded by the gene isolated from the marine copepod Gaussia princeps. This luciferase, which does not require ATP, catalyzes the oxidation of the substrate coelenterazine (Product M0739) in a reaction that emits light (at 470 nm), and has considerable advantages over other luciferases. Gaussia luciferase has a natural secretory sequence and is therefore secreted into the cell medium of transfected cells grown in culture, making cell lysis unnecessary for assaying expression levels. In addition, Gaussia luciferase is reported to generate over 1000-fold higher bioluminescent signal intensity compared to Firefly or Renilla Luciferases. Recently, researchers from the laboratory of Dr. Theodore K. Cristopoulos at The Department of Chemistry and Biochemistry, University of Windsor, have cloned a modified version of this luciferase that includes a biotin acceptor peptide as a fusion protein with the GLuc as well as a biotin protein ligase (BPL). This second element catalyzes the covalent attachment of a single biotin to the final GLuc fusion protein in vivo. Purification of the GLuc luciferase can then be accomplished by simple affinity chromatography. Besides facilitating purification, the in vivo biotinylated luciferase can then be used for determination of DNA by using a sandwich microtiter well-based DNA hybridization assay method in which DNA is coated onto the wells of plates and hybridized to a biotinylated DNA probe. After washing steps, the hybridized DNA is detected by adding streptavidin, followed by the new biotin-GLuc and coelenterazine. The resulting chemiluminescence exhibited a linear relationship to DNA concentration over a wide range, with good reproducibility, although autooxidation of coelenterazine during these assays was somewhat problematic. For more information about these new bioluminometric systems, please see the references below, or visit our website.

• Monique Verhaegen Theodore K. Christopoulos (2002) "Recombinant Gaussia Luciferase. Overexpression,
  Purification, and Analytical Application of a Bioluminescent Reporter for DNA Hybridization." Anal. Chem. 74: 4378-4385.
• Bakhos A. Tannous, Eleftheria Laios and Theodore K. Christopoulos (2002) "T7 RNA polymerase as a self-replicating label for antigen quantification" Nucleic Acids Research, 2002, 30(24): e140.
• Laios E, Obeid PJ, Ioannou PC, Christopoulos TK.(2000) "Expression hybridization assays combining cDNAs from firefly and Renilla luciferases as labels for simultaneous determination of two target sequences." Anal Chem. 72(17):4022-8.
• Laios E, Ioannou PC, Christopoulos TK. (2001) " Enzyme-amplified aequorin-based bioluminometric hybridization assays"
  Anal Chem.;73(3):689-92.

Ethidium Homodimer (EthD-1)

Ethidium Homodimer (M1093) is a high affinity fluorescent nucleic acid probe that binds to both DNA and RNA in a sequence-independent manner. Upon binding there is a >30-fold fluorescence enhancement. The DNA binding of each Ethidium Homodimer covers four base pairs and is believed to occur by intercalation. Because the dye is highly positively charged, it cannot cross cell membranes to stain living cells. It is therefore widely used as a viability probe and will only stain dead, damaged or injured cells whose membranes have been compromised and allow the stain to penetrate. The typical staining protocol is to incubate live cell monolayers in the dark at 37°C with 0.6 -2.0 mM Ethidium Homodimer-1 (M1093) for 15 min and detect the red fluorescence at or near 630 nm with excitation at 530 nm. Often, a long pass filter can be used to allow the detection of several stains (for viability assays) concurrently. For more information about these techniques, please see the references below, or visit our website.

• A. Louise McCormick, Christopher D. Meiering, Geoffrey B. Smith, and Edward S. Mocarski "Mitochondrial Cell Death Suppressors Carried by Human and Murine Cytomegalovirus Confer Resistance to Proteasome Inhibitor-Induced Apoptosis" J Virol. 2005 October; 79(19): 12205–12217.
• Karen L. Oslund, Lisa A. Miller, Jodie L. Usachenko, Nancy K. Tyler, Reen Wu and Dallas M. Hyde "Oxidant-Injured Airway Epithelial Cells Upregulate Thioredoxin but Do Not Produce Interleukin-8" American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 597-604, 2004

In vitro Luciferin-Galactoside assays.

Both ß-Galactosidase and luciferase are widely used as a marker genes for measuring gene expression levels, for protein-protein interaction studies based on the yeast two-hybrid enzyme complementation system as well as for antibody conjugate staining in cell sorting, monitoring and counting. One of the most sensitive detection substrates available to measure b-Gal activity has become the chemiluminescent compound, D-Luciferin-6-O-b-D-Galactoside (M1087) (Luc-Gal). When used as a coupled enzyme reaction, where added firefly luciferase is used to develop the light signal, picomolar concentrations of analyte can be measured, without washing steps, in a microplate assay format.  Marker Gene provides this high-purity, ultrasensitive substrate in a convenient lyophilized form. In the example above, a combined ß-Gal:Luciferase enzyme sample was prepared in our M0626 reaction buffer containing 2mM ATP and 1mM M1087. We found that M1087 is very soluble in water with very low background when using only luciferase (no ß-Gal). With much higher luciferase concentrations (>1 million units/ml ), some background was noticed but ß-Gal-containing samples had readings >Max and the light emission was even visible to naked eye. For more information about these assays and the new Luc-Gal substrate, please see the references below, visit our website, or contact us for more help with the development of your assay methods and systems.

• Henderson, D.R. Friedman, S.B. Harris, J.D. Manning, W.B. Zoccoli, M.A., (1986)”CEDIA, a new homogeneous immunoassay system” Clin.Chem.32: 1637 –1641.
• Yang,Y., Janatova, J., Andrade, J.D., (2005) “Homogeneous enzyme immunoassay modified for application to luminescence-based biosensors” Anal. Biochem. 33: 102 –107.
• Khanna, P.L. Dworschack R.T., Manning W.B., Harris J.D.,  (1989) “A new homogeneous enzyme immunoassay using recombinant enzyme fragments.” Clin. Chim. Acta. 15: 231–9.
• Geiger R.,  Schneider E.,  Wallenfels K.,  Miska W., (1992)  “A new ultrasensitive bioluminogenic enzyme substrate for beta-galactosidase”     Biol. Chem. Hoppe-Seyler  373(12): 1187-91.
• Ugarova, N. N.; Voznyi, Ya. V.; Kutuzova, G. D.; Dement'eva, E. I.   (1991) “Bioluminescent assay of b-galactosidase using D-luciferin-o-b-galactoside.” Biolumin. Chemilumin. Proc. Int. Symp., 6th  (1991), pp. 511-14.  Publisher: Wiley Intersceince, Chichester, UK  Editor(s): Stanley, PE.; Kricka, LJ. 
• Wehrman TS , von Degenfeld G , Krutzik PO , Nolan GP , Blau HM “Luminescent imaging of beta-galactosidase activity in living subjects using sequential reporter-enzyme luminescence.” Nature methods 3(4): 295-301.
• Geiger R , Schneider E , Wallenfels K , Miska W  (1992) “A new ultrasensitive bioluminogenic enzyme substrate for beta-galactosidase.” Biological chemistry Hoppe-Seyler  373(12): 1187-91.
  

Peptidase Substrates from 6-FMOC-Amino-D-Luciferin.

6-FMOC-Amino-D-Luciferin (M0802) is a amino-protected derivative of the firefly luciferase substrate 6-amino-D-luciferin (M0352) that can be used in automated peptide synthesis for production of luciferin-conjugated peptides. Many bioluminescent protease assays have been reported using conjugated aminoluciferin peptidase substrates. These studies have demonstrated that once a peptide is conjugated to aminoluciferin it is no longer a substrate for luciferase and will not produce light. It is not until the peptide is cleaved by its specific peptidase activity and the aminoluciferin released that light emission will occur. In these reports, the protease is typically first incubated with the peptide-aminoluciferin substrate, and the released aminoluciferin is allowed to accumulate. Then a sample of this reaction product is incubated with luciferase to generate light emission at 560 nm in direct proportion to the amount of peptidase activity present in the first sample. These assays can be incorporated into a high-throughput system for homogeneous, bioluminescent protease assays that are comparable in simplicity to existing fluorescent protease assays but exhibit the high sensitivity of bioluminescence detection. For more information about this exciting new synthon for making new peptidase substrates, please visit our website or see the references below.

• "Transmembrane motility assay of transiently transfected cells by fluorescent cell counting and luciferase measurement." Gildea JJ, Harding MA, Gulding KM, Theodorescu D. Biotechniques 29(1): 81-86 (2000).
• "The use of the luciferase reporter system for in planta gene expression studies." Van Leeuwen W, et al. Plant Mol. Biol. Rep. 18: 143a (2000).
• "Amino Analogs of Firefly Luciferin and Biological Activity Thereof." White, E.H., Worther, H., Seliger, W.D., J. Amer. Chem. Soc. (1966) 88:2015-2019.
• "Chemiluminescent and bioluminescent reporter gene assays." Bronstein I, Fortin J, Stanley PE, Stewart GS, Kricka LJ. Anal. Biochem. 219(2): 169-181 (1994).
• "Firefly luciferase, synthesized to very high levels in caterpillars infected with a recombinant baculovirus, can also be used as an effective reporter enzyme in vivo." Jha PK, Nakhai B, Sridhar P, Talwar GP, Hasnain SE. FEBS Lett. 274(1-2): 23-26 (1990).
• "Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants." Ow DW, et al. Science 234: 856 (1986).
• Monsees T, Miska W, Geiger R: Synthesis and characterization of a bioluminogenic substrate for alpha-chymotrypsin. Anal Biochem 1994;221:329-334.
• Monsees T, Geiger R, Miska W: A novel bioluminogenic assay for alpha-chymotrypsin. J Biolumin Chemilumin 1995;10:213-218.
• Niles A, Moravec R, Scurria M, O’Brien M, Riss T: A novel homogeneous bioluminescent caspase-8 activity assay [Poster 274]. Paper presented at the Keystone Symposia: Molecular Mechanisms of Apoptosis, Banff, Alberta,
  February 2003
• See also our WebNewsletter www.markergene.com/WebNewsletter6.5.htm

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Marker Gene strives to offer our customers products of the highest quality and at the best possible prices.  Our years of experience allow us to provide timely products for less cost to you.  See our latest Price Comparison Chart that compares our prices with those from several alternate sources, to see if you can save money by switching to Marker Gene (http://www.markergene.com/crossref.htm).  Or visit our website at www.markergene.com and click on the link “COMPARE”.  We think you will appreciate our efforts to keep costs low and maintain excellent quality of our products for your research.  For more information about any of our products, simply telephone us toll free at 1-888-218-4062 or contact us by e-mail at techservice@markergene.com.  We will be happy to send you more about our products and their specifications.

CONTRACT  RESEARCH@markergene.com

Marker Gene Technologies, Inc. has the expertise to perform contract research with you on your project. We have worked with many biotechnology and pharmaceutical companies on successful, proprietary and patented projects.

Contract Research and Development Capabilities in the following areas:

• Established in 1993 at the University of Oregon Riverfront Research Park.
• Screening Assay Development for HTS and uHTS
• Chemical and Cellular Assays – High-Content Screening.
• DNA/RNA (genomics) and protein (proteomics) labeling and assay development.
• Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture.
• Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.
• Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical).
• Confidentiality, help in patent preparation and filings.

Contact us by telephone at (888) 218-4062 or (541) 342-3760 or FAX us at (541) 342-1960 or you can write to us at  Contract Research, Marker Gene Technologies, Inc., 1850 Millrace Drive, Eugene, Oregon 97403-1992 or contact us by e-mail at: techservice@markergene.com

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