Marker Gene Monthly Newsletter   

March, 2007

Volume 7, Number 3

© 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.

His6-Tag and Fluorescent Nitrilotriacetates (NTAs)
for Binding and Measuring Membrane Proteins.

Many methods have been developed to monitor native and recombinant proteins in living cells.  Tese include chemical synthesis with modification of amino acid side chains, biosynthetic fusion with GFP or other fluorescent proteins, genetic fusion with enzymes that can turn over fluorescent substrates, fusion with protein sequences that are capable of covalent labeling and affinity labeling with fluorescent antibodies or lectins.  Each of these methods has specific advantages that can be used to help with monitoring protein levels or trafficking in cells or tissues.  Such chemical or biological labeling has been fundamental hin elucidating the function of proteins within biochemical cellular networks. In particular, fluorescent probes have allowed detection of molecular interactions, mobility and conformational changes of proteins in live cells.  A new and interesting method of labeling and/or purifying recombinant proteins has been developed that utilizes an oligohistidine sequence (hexahistidine, His6) tag as a recognition element for site-selective labeling incombination with a metal-ion-chelating nitrilotriacetate (NTA) compound.  The nitrilotriacetate (NTA) group selectively and stably binds to the oligohistidine tag through multiple ionic interactions.  If the NTA is further linked to a fluorophore, the small NTA-fluorophore probe can be used to directly label the His6 tagged protein. The His6 tag must, however, be genetically encoded into a region of the amino acid sequence that will not disturb the protein's structure or function.  Typically it is located in terminal regions of the protein or in protein loops.  The tags are not tolerated within structurally important regions such as helical membrane spanning sections of proteins, and often using C-terminal or N-terminal sites will have different binding and activity effects.  Nevertheless, the His6 tag sequences have been applied in combination with NTA-linked molecules or columns for purification, for in vitro detection or for surface labeling of recombinant proteins.  Because the NTA-fluorophore probes are highly charged, they do not freely pass through the cell membrane and are typically used for measuring membrane protein systems.  NTA-fluorophore conjugates containing several rhodamine and fluorescein labels have been prepared, and vectors containing the His6 tag have become commercially available.   For more information about these new labels and their uses, please visit our website or see the references below. 

• Tropea JE,  Cherry S, Nallamsetty S, Bignon C, Waugh DS  (2007) “A generic method for the production of recombinant proteins in Escherichia coli using a dual hexahistidine-maltose-binding protein affinity tag.” Meth. Mol. Biol.  363: 1-19.
  
• Guignet EG , Hovius R , Vogel H (2004) “Reversible site-selective labeling of membrane proteins in live cells.” Nature Biotechnology  22(4): 440-4.
  
•  Kiefer H , Vogel R , Maier K (2000) “Bacterial expression of G-protein-coupled receptors: prediction of expression levels from sequence.”  Receptors & Channels 7(2): 109-19.
  
•  Hochuli, E., Dobeli, H. & Schacher, A. New metal chelate absorbant selective for proteins and peptides containing neighbouring histidine residues. J. Chromatography 411, 177–184 (1987).
  
• Stora, T., Hovius, R., Dienes, Z., Pachoud, M. & Vogel, H. Metal ion trace detection by a chelator-modified gold electrode: a comparison of surface to bulk affinity. Langmuir 13, 5211–5214 (1997).
  
• Holmes, K.L. & Lantz, L.M. Protein labeling with fluorescent probes. Methods Cell Biol. 63, 185–204 (2001).
  
• Keppler, A., Gendreizig, S., Gronemeyer, T., Pick H., Vogel H., Johnsson  K., A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).
  
• Griffin, B.A., Adams, S.R. & Tsien, R.Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).
  
• Giriat, I, Muir, T.W. Protein semi-synthesis in living cells. J. Am. Chem. Soc. 215, 7180–7181 (2003).
  
•  McMahan, S.A. & Burgess, R.R. Single-step synthesis and characterization of biotinylated nitrilotriaceticacid, a unique reagent for the detection of histidine-tagged proteins immobilized on nitrocellulose. Anal. Biochem. 236, 101–106 (1996).
• Kapanidis, A.N., Ebright, Y.W. & Ebright, R.H. Site-specific incorporation of fluorescent probes into protein: hexahistidine-tag-mediated fluorescent labeling with (Ni(2+):nitrilotriacetic acid (n)-fluorochrome conjugates. J. Am. Chem. Soc. 123, 12123–12125 (2001).

Ultrasensitive lacZ Detection in vivo using
D-Luciferin-6-O-Galactopyranoside (Luc-Gal).

b-Galactosidase (lacZ) and firefly luciferase are two of the most widely used marker genes for measuring gene expression levels, for promoter activity analysis, to measure protein levels or mobility through fusion proteins, or for use in protein-protein interaction studies based on the yeast two-hybrid enzyme complementation assays, as well as many other applications.  Recently, the combined ultrasensitive detection substrate, D-Luciferin-6-O-b-D-Galactopyranoside (Luc-Gal, M1087) (also termed Beta-Glo®) has been used for in vivo analysis of lacZ beta-galactosidase activity in a variety of systems.  This substrate, a caged D-luciferin-galactoside conjugate, must first be cleaved by beta-galactosidase before it can be catalyzed by firefly luciferase (luc) to generate light.  As a result, luminescence becomes dependent on lacZ expression and activity.  Using this substrate, methods have been developed to monitor expressed beta-galactosidase levels in transgenic lacZ cell lines as well as inducible tissue-specific lacZ expression in vivo, noninvasively and without incident light sources for illumination.  Another advantage of using lacZ beta-galactosidase as a bioluminescent probe is that this enzyme does not require ATP or other cofactors for extracellular enzyme detection methods, in contrast to luciferase which requires such intracellular cofactors.  As a result, antibodies conjugated to the beta-galactosidase enzyme can be used to detect specific cells or tissues through extracellular antigens in vivo.   Coupling of the ultrasensitive chemiluminescent detection properties of firefly luciferase system to the advantages of lacZ beta-galactosidase permits bioluminescent imaging applications that were previously not obtainable by other means.  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 information.

• 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),  Meeting Date 1990,   pp. 511-14.  Publisher: Wiley Intersceince, Chichester, UK  Editor(s): Stanley, Philip E., Kricka, Larry J.  
• 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.
• 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),  Meeting Date 1990,   pp. 511-14.  Publisher: Wiley Intersceince, Chichester, UK  Editor(s): Stanley, Philip E., Kricka, Larry J. 
• Beta-Glo®is a registered trademark of Promega, Corporation (Madison, WI). 

Esterase Assays as a Measure of Cell Viability.

Marker Gene provides several essential substrates that can be used for such viability and cell biology assays.  These include 5(6)-Carboxyfluorescein diacetate (CFDA)(M0011), Fluorescein diacetate (FDA) (M0060), 2, 7-Dichlorofluorescin Diacetate (M0807), Rose Bengal Diacetate (M0780) and 3-(2-Benzoxazolyl)umbelliferyl acetate (M1253).  Several of these fluorogenic substrates have secondary detection properties (singlet oxygen, ROS detection, etc.)   In addition, our sensitive Marker GeneTM Live:Dead Assay Kit (M0795) can be used with mammalian cells, bacteria and yeast cells in culture to provide a quick, accurate and quantitative analysis of cell viability using the two vital stains, carboxyfluorescein di-acetate (M0011) and propidium iodide (M0793) in a protocol that allows staining of up to 1000 microplates of cells.  Analyses by fluorescence microscopy, flow cytometry or by using standard microtiterplate formats are easily performed and the red and green fluorescence emissions are readily resolved.  The determination of live and dead cells can be measured by intracellular esterase activity and plasma membrane integrity. Carboxyfluorescein di-acetate and propidium iodide are optimal dyes for this application. Assessing cell viability with this fluorescence-based method can replace similar methods for determining cell cytotoxicity and viability including trypan blue exclusion, and 51Cr release, which tend to be slower, more expensive, and less sensitive indicators of cytotoxic events.  The enzymatic conversion of the cell-permeant, non-fluorescent carboxyfluorescein diacetate to the intensely fluorescent carboxyfluorescein is an indicator of intracellular esterase activity, a characteristic of live cells. The carboxyfluorescein dye is retained within live cells, producing a green fluorescence, with excitation and emission at ~475nm and 517nm, respectively. Cells with damaged membranes allow the entrance of propidium iodide, which undergoes a fluorescence enhancement upon binding to nucleic acids, promoting a red fluorescence in dead cells with excitation and emission at ~493nm and 630nm, respectively. The intact plasma membrane of live cells excludes propidium iodide. Determination of cell viability with this kit depends on these specific biochemical properties; cytotoxic events having no effect on these properties might not be accurately measured by this method. With this assay technique, background fluorescence levels are inherently low because before interacting with cells, the dyes are virtually nonfluorescent. For more information about this assay, see the references below.

• Jones KH, Senft JA, (1985) “An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide” J. Histochem. Cytochem. 33: 77
• Bottiroli G, Croce AC, Balzarini P, Locatelli D, Baglioni P, Lo Nostro P, Monici M, Pratesi R. (1997) “"Enzyme-assisted cell photosensitization: a proposal for an efficient approach to tumor therapy and diagnosis. The rose bengal fluorogenic substrate." Photochem. Photobiol. 66: 374-379.
• Kroesen, B.J., Mesander, G., ter Haar, J.G., The, T.H., de Leij, L., (1992)  "Direct visualisation and quantification of cellular cytotoxicity using two colour flourescence." J. Immunol. Methods 156:47-54.
• Zurgil, N., Shafran, Y., Fixler, D., Deutsch, M., (2002) "Analysis of early apoptotic events in individual cells by fluorescence intensity and polarization measurements." Biochem. Biophys. Res. Commun. 290:1573-1582.
• Bunthof, C.J., Bloemen, K., Breeuwer, P., Rombouts, F.M., Abee, T., (2001) “Flow cytometric assessment of viability of lactic acid bacteria." Appl. Environ. Microbiol. 67:2326-2335
•  Deere D, Shen J, Vesey G, Bell P, Bissinger P, Veal D. (1998)"Flow cytometry and cell sorting for yeast viability assessment and cell selection." Yeast 14, 147-160
•  Chang, L., Gusewitch, G.A., Chritton, D.B., Folz, J.C., Lebeck, L.K., Nehlsen-Cannarella, S.L., (1993) "Rapid flow cytometric assay for the assessment of natural killer cell activity." J. Immunol. Methods. 166:45-54.
•   Pec, M.K., Aguirre, A., Fernandez, J.J., Souto, M.L., Dorta, J.F., Villar, J.. "Dehydrothyrsiferol does not modulate multidrug resistance-associated protein 1 resistance: a functional screening system for MRP1 substrates." Int J. Mol. Med. 10:605-608.
• Riordan, H.D., Riordan, N.H., Meng, X., Zhong, J., Jackson, J.A., (1994) "Improved microplate fluorometer counting of viable tumor and normal cells." Anticancer Res. 14:927-931.

Fluorescent Heparin Sulfate Measurement with Berberine. 

Heparin and heparan sulphate are important biological polysaccharides that both have the same basic structure consisting of repeating disaccharides of D-glucuronic acid and N-acetylgalactosamine of about 50K MW.  Heparin is widely known for its anti-coagulant activity, based on its binding with antithrombin III.   The molecular differences between heparin and heparan sulphate are minor.   They both contain numerous variations of sulphonation.  The amount of N-sulfation has occasionally been used to make distinction between heparin and heparan sulphate so that in heparan sulphate the proportion of N-sulfation is below 50% while in heparin it is usually above 70%.  Heparin has been utilized in a variety of biological assays including inhibition of vascular smooth muscle cell proliferation and migration both in cell culture and in animal models, inhibition of amyloidogenesis in cell culture (by both native heparin (Mr 12,000) and low molecular weight heparin (Mr 3000), as well as stimulation of collagenase (matrix metalloproteinase-1) synthesis in histiotypic epithelial cell culture.  It has also recently been implicated in the clearance of triglyceride-rich lipoproteins independently of LDL in vivo in the liver.  It has even found use as a substrate for stimulating Staphylococcus aureus biofilm formation. 

The weakly fluorescent cationic dye, berberine sulfate (M1257) forms a strongly fluorescent complex with the heparin and has been used for cytofluorometric measurement of heparin in a variety of cell lines, including mast cells and individual mast cell granules. Berberine can be used as a vital stain, demonstrating the secretory activity of mast cells. At a dye concentration of 0.025%, and after membrane stabilization with polyethylene glycol, normal mast cells exclude the dye while mast cells stimulated to secretion with polymyxin B show a strongly fluorescent dye binding to individual cytoplasmic granules. The mean fluorescence intensity (reflecting the number of stained granules) of the cell populations increases with increasing polymyxin B concentrations up to 2.0 micrograms/ml, thereafter remaining constant up to 10 micrograms/ml.  Fluorescence intensity after vital staining with berberine was compared both to release of heparin measured by berberine binding to fixed cells (reflecting exocytosis of mast cell granules) and also to histamine release.  For measurement of heparin, cells are fixed with 5% glacial acetic acid in ethanol for 60 min, and then stained with a solution of  0.02% berberine sulfate in water adjusted containing citric acid to pH 4.0 for 20 min.  The cells are then examined by fluorescence microscopy.   For more information about heparin assays and the use of berberine for heparin detection, please see the references below or visit our website. 

• Dimlich RV, Meineke HA, Reilly FD, McCuskey RS (1980) “The fluorescent staining of heparin in mast cells using berberine sulfate: compatibility with paraformaldehyde or o-phthalaldehyde induced fluorescence and metachromasia.” Stain Technol. Jul;55(4):217-23.
• Stermitz, F.R., (2000) “Synergy in a medicinal plant: Antimicrobial action of berberine potentiated by 5'-methoxyhydnocarpin, a multidrug pump inhibitor.” Proc. Natl. Acad. Sci. USA 97: 1433.
• Berlin, G., Enerback, L. (1984) “Non-differential inhibition of histamine and serotonin release from mast cells by amitriptyline.” Agents and Actions 14: 401.
• Lin, H.L. (1999) “Up-regulation of multidrug resistance transporter expression by berberine in human and murine hepatoma cells” Cancer 85: 1937-1942.
• Schumacher, M.A., Miller, MC, Grkovic, S,  Brown, MH,  Skurray, RA, Brennan RG, (2001) “Structural Mechanisms of QacR Induction and Multidrug Recognition “ Science, 294: 2158-2163.
• Enerback L. (1974)  “Berberine sulphate binding to mast cell polyanions: A cytofluorometric method for the quantitation of heparin.” Histochemistry 42: 301–313.

Compare Our Quality. 

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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