Wilchek M. Et
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Meir Wilchek Recent advances in the Edward A. Bayer Heike Hofstetter avidin-biotin-system Oliver Hofstetter Tikva Kulik Margherita Morpurgo Department of Biological Chemistry Tel. +972 8 934 3808 Fax. +972 8 946 8256 E.mail: [email protected] Novel nonradioactive labeling and detection This principle was demonstrated using avidin, which binds systems the two ligands biotin and HABA with different affinities. Nonradioactive labeling reagents are of great interest Avidin was affinity labeled with an activated, cyclic in bioanalytical applications, e.g., for detection, derivative of HABA. Due to the properties of the cyclic localization, isolation and purification purposes. Existing reagent four HABA-molecules were bound to the binding systems suffer from several disadvantages, including sites of avidin, one per subunit. Antibodies raised against difficulties in determining the amount of label and HABA did not bind HABA buried in the binding site of problems in separating labeled from unlabeled molecules. avidin. However, upon addition of biotin HABA was We decided to introduce a new label that is easily detected expelled from the binding site and immediately bound by and can be conjugated to all kinds of biomolecules, thus the antibodies (Fig. 2). In this way multilayer assemblies providing universal applicability (Hofstetter et al., in of HABAylated avidin and biotinylated anti-HABA preparation). A number of derivatives of the dye 4- antibodies could be constructed. hydroxyazobenzene-2-carboxylic acid (HABA) was Biotin-saturated HABAylated avidin was incubated synthesized (Fig. 1) and the terminal functional groups of with biotinylated anti-HABA antibodies, followed by the tag were activated to enable covalent attachment to additional cycles of HABAylated avidin and biotinylated biomolecules. antibodies. A stepwise increase in absorbance could be Various proteins and peptides were labeled and the detected after the formation of each layer. The cascade amount of label was determined spectrophotometrically could be initiated using HABAylated avidin and biotin or λ ≈ using the characteristic yellow color of HABA ( max biotinylated macromolecules. In either case, the addition 350 nm). The modified molecules could be separated of biotin was crucial to expel the HABA from the binding from their native, unmodified form by adsorption on an site, thus enabling subsequent interaction with the anti- avidin column followed by elution under mild conditions HABA antibodies due to the moderate affinity of HABA for avidin This concept may find application in numerous fields, ≈ -6 (Kd 10 M). HABAylated proteins and peptides were such as medicine, diagnostics and nanotechnology. It also detected using anti-HABA antibodies modified with mimics by analogy how signals are being transduced in different probes. The new, nonradioactive, colored nature via cascades of proteins changing low affinity labeling reagents based on HABA will add new dimensions interactions to high affinity interactions after ligand and expand the scope of existing systems. binding. Avidin-HABA (red avidin) Novel and useful derivatives of avidin A new principle to construct organized systems, based prepared by genetic engineering on competing affinities between two different ligands for Despite the usefulness of avidin in many applications of the same binding site of a protein, was developed using avidin-biotin technology, the high pI of avidin and the an avidin-HABA conjugate (Morpurgo et al., 1998). If presence of carbohydrate can cause nonspecific binding the lower affinity ligand is attached covalently to the binding site of the protein via an appropriate spacer, it will be prevented to interact with other proteins that recognize it. However, if the higher affinity ligand or a molecule containing this ligand is added, it will displace the low affinity ligand from the binding site to the periphery but still remain covalently bound. The low affinity ligand is now available for interaction with other molecules. This provides the means to assemble multilayers of proteins by a recognition cascade. Fig. 1. HABA-analogues used for the preparation of derivatives. 238 capable of binding biotin and biotinylated material. Their binding affinities to immobilized biotin matrices was reduced and could be reversed by free biotin. Both W→K mutants were found to form stable dimers in solution. The W→K mutants appear to represent the long-sought- after avidin variants that can be used for reversible, affinity- based isolation of biotinylated material. References Laitinen, O.H., Airenne, K.J., Marttila, A.T., Kulik, T., Porkka, E., Bayer, E.A., Wilchek, M., Kulomaa, M.S. Mutation of a critical tryptophan to lysine in avidin or streptavidin may explain why sea urchin fibropellin adopts an avidin-like domain. FEBS Lett. , 461, 52-58 Marttila, A.T., Airenne, K.J., Laitinen, O.H., Kulik, T., Bayer, E.A., Wilchek, M., Kulomaa, M.S. (1998) Engineering of chicken avidin: a progressive series of reduced charge mutants. FEBS Lett. 441, 313-317. Morpurgo, M., Hofstetter, H., Bayer, E.A., and Fig. 2. Red avidin, λ = 504 nm, does not interact with anti-HABA max Wilchek, M. (1998) A chemical approach to illustrate antibody. The cascade is triggered upon addition of biotin (B) which the principle of signal transduction cascades using the expels the HABA moiety from the binding site (shift to orange, λmax = 356 nm) rendering it available for subsequent interaction with anti-HABA avidin-biotin-system. J. Am. Chem. Soc. 120, 12734- antibody or avidin 12739. Acknowledgements thus hindering its use. Streptavidin, a nonglycosylated M. Wilchek holds the Marc R. Gutwirth Professorial and neutrally charged bacterial counterpart of avidin, has Chair in Molecular Biology. The research was supported therefore replaced avidin in such applications. In attempts by the Israel Academy of Science and Humanity and by to improve the molecular properties of avidin, mutated MINERVA, Deutsch-Israelische Wissenschaftliche forms were cloned and expressed in a baculovirus-infected Zusammenarbeit, Germany. insect cell system. Using this approach, we demonstrated that the high pI of avidin can be reduced down to 4.7 without losing its biotin-binding capacity or the stability of the avidin tetramer (Marttila et al., 1998). In further work (Marttila et al., in preparation), we changed the glycosylation site of avidin in order to produce a recombinant form of avidin which lacks the carbohydrate moiety to further improve the non-specific binding characteristics of avidin. The resultant neutrally charged, sugarless mutant of avidin displayed strong biotin-binding activity and reduced non- specific binding to DNA and different cells, thus offering new possibilities for application in avidin-biotin technology. In another direction, a distant relative of avidin, sea-urchin fibropellin, was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin- binding sites of avidin and streptavidin (Laitinen et al., 1999). Based on the fibropellin sequence, an intriguing mutant of avidin (Trp110Lys) was designed and expressed. A related streptavidin mutant (Trp120Lys) was similarly expressed. The homologous tryptophan-to-lysine (W→K) mutations of avidin and streptavidin were both 239.