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Protein Science (2004), 13:3066-3070. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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AWARD ADDRESS

My life with affinity

Meir Wilchek1

Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel


   Introduction
TOP
 Introduction
Scientific life
 Affinity chromatography
 Affinity labeling
 Affinity therapy
 The avidin-biotin system
 Finale
 References
 
I was born in 1935 in Warsaw, Poland, to a rabbinical family. My early childhood occurred during World War II in German-occupied Poland and then in Russia. My father was killed in the concentration camp of Flossenberg; my mother, my sister and I survived and emigrated from Germany to Israel in 1949. In Israel I finished my primary school and high school. I completed my B.Sc. in chemistry and physics at Bar-Ilan University and my Ph.D. in biochemistry at the Weizmann Institute of Science.


   Scientific life
TOP
 Introduction
 Scientific life
Affinity chromatography
 Affinity labeling
 Affinity therapy
 The avidin-biotin system
 Finale
 References
 
Most of my scientific career has been devoted to using chemistry to investigate biological recognition and the application of the phenomenon of biorecognition to solve biologically important problems. I have always tried to maintain an equilibrium at the interface between chemistry and the life sciences.

During the years, my efforts have mainly been devoted to different but related directions: namely, affinity chromatography, affinity labeling, affinity therapy, and the avidin–biotin system—the highest affinity interaction—which combines all of the former approaches. But, before I start to describe the above, I would like to mention some early contributions of mine, which formed the foundation for establishing my work at the chemistry–biology interface. These include the conversion of serines to cysteines in peptides (Zioudrou et al. 1965), which was applied later to proteins, my involvement with energy transfer studies between aromatic amino acid chromophores (an approach known today as FRET; Edelhoch et al. 1967), and the fine structure of these chromophores using circular dichroism (Edelhoch et al. 1968). However, my major contribution has been in the development and application of biorecognition, to which I will devote the rest of the space allocated to me.


   Affinity chromatography
TOP
 Introduction
 Scientific life
 Affinity chromatography
Affinity labeling
 Affinity therapy
 The avidin-biotin system
 Finale
 References
 
Affinity chromatography is a method in which biospecific and reversible interactions are used for the selective extraction, separation, and/or purification of biologically active material from crude samples (Fig. 1Go). The approach was introduced in 1968 by Cuatrecasas, Anfinsen, and myself to purify proteins (Cuatrecasas et al. 1968), and today it still represents one of the most powerful techniques available for purifying of biologically active compounds. The method is also an indispensable tool for studying many biological processes, such as the mechanism of action of enzymes, hormones, protein–protein or cell–cell interactions and others. In the initial two demonstrations of this approach, we immobilized biotin to purify avidin and thymidine phosphate to purify nuclease. In both cases, the polymeric carrier was a polysaccharide, Sepharose, and the reaction used to immobilize the ligand was cyanagen bromide. In order to increase the efficacy of purification, we also introduced the principle of spacers between the biologically active ligand and the inert polymer. Interestingly, even today 95% of all affinity purification methods apply the same general principles outlined by us (see Fig. 1Go for a short list of types of biomolecules purified by affinity chromatography).



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  		Figure 1. Scheme showing principle of affinity chromatography and short list of target materials that have been purified.

 
Following the initial introduction of affinity chromatography and upon returning to Israel I continued to work in this field, and, in doing so, my group continued to furnish many novel contributions. We continued to develop new carriers and new methods of ligand coupling. All of these carriers and coupling methods were immediately adopted by many research facilities as well as biotechnology-based industries worldwide. We also developed quantitative analytical methods for determining the extent of activation and amount of bound ligand. We also established the chemical mechanism of these reactions. These studies were initiated to solve many problems that are inherent in the technique of affinity chromatography, such as ligand leakage, hydrophobic interactions, and ion-exchange problems. The information gained through these studies has enabled us to develop new approaches for coupling proteins and ligands to solid matrices in a leakage-free manner. In addition, we were able to explain the forces involved in hydrophobic chromatography, which has in turn helped us to develop even more efficient affinity columns (Wilchek et al. 1984).

The chemical nature of this work has not detracted from our continued application of these columns for the isolation and study of biologically active molecules. For example, in 1971 we showed for the first time, using a cAMP-containing affinity column, that protein kinase is composed of regulatory and catalytic subunits (Wilchek et al. 1971b). We were also the first to employ antibody columns for the isolation of antigenic peptides derived from parent proteins (Wilchek et al. 1971a). Immunoaffinity chromatography continues to be a definitive tool for the isolation of proteins produced by genetic engineering. We also showed that affinity columns can be used to remove toxic compounds from blood, as exemplified by the removal of heme peptides from blood using immobilized human serum albumin, thus laying the grounds for modern-day hemoperfusion (Wilchek 1972). Moreover, we introduced the concept of sandwich-type affinity chromatography, whereby either an antibody to a hapten or avidin was used to isolate a biologically active molecule via the hapten-modified or biotinylated counterpart to a target molecule.

The general biorecognition-based approach was subsequently adopted for a variety of other techniques (Table 1Go). Affinity chromatography is thus the grandfather of most modern techniques, including biosensors, DNA, and protein microarrays, and their varied application in diagnostics, protein–protein interactions and drug screening.


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  		Table 1. Various techniques derived from Affinity Chromatography
 

   Affinity labeling
TOP
 Introduction
 Scientific life
 Affinity chromatography
 Affinity labeling
Affinity therapy
 The avidin-biotin system
 Finale
 References
 
Concurrent with affinity chromatography I continued to work in areas in which I could combine chemical reactions with the affinity concept to investigate the phenomenon of biological recognition. A perfect example of such an approach is affinity labeling, by which residues in the binding-active site of proteins can be identified. Using this technique, we determined which amino acid residues are involved in the active sites of enzymes (such as nuclease; Cuatrecasas et al. 1969). In the field of antibodies, we were able to prove that their binding site lies in the Fv portion of the molecule and involves three hypervariable sites, today called the complementarity-determining regions (CDRs; Strausbauch et al. 1971). The latter work was instrumental in the preparation of X-ray models of the antibody molecule and for the preparation of Fv, which is very popular today, and which we synthesized chemically as early as 1977 (Gavish et al. 1978). We were also the first to affinity label and photoaffinity label complex molecules, e.g., ribosomes and receptors in the intact cell (Wilchek 1984). These early studies, which were published in the early 1970s, were verified more recently.

With the advancement of X-ray crystallography, the importance of affinity labeling as a general tool to study binding sites has diminished, but it is still used for crosslinking betwen biologically active partners and for localization of drug targets and for nanobiotechnology as we have shown recenty (Morpurgo et al. 1998).


   Affinity therapy
TOP
 Introduction
 Scientific life
 Affinity chromatography
 Affinity labeling
 Affinity therapy
The avidin-biotin system
 Finale
 References
 
Affinity therapy is a biorecognition-based approach to selectively deliver a cytotoxic drug or toxin to a given target cell located where the drug is needed. The cell-associated target molecule can be a defined cell-surface antigen, a surface receptor or other type of biomolecule which bears specificity for a given antibody, hormone, or nutrient. The drug counterpart can comprise the corresponding antibody, hormone, etc. to which a cytotoxic compound (e.g., selected from chemotherapeutic drugs, radionucleotides, or toxins from different origins) has been chemically attached. We called this approach by a general term, affinity therapy, which was later changed by others to the misnomer, immunotoxins.

In the field of affinity therapy, which was pioneered together with Michael Sela, Ester Hurwitz, and Ruth Arnon as early as 1975 (Hurwitz et al. 1975), we applied drug-conjugated antibodies for the targeted delivery of cytotoxic compounds to cancer cells. We developed methods to conjugate drugs and toxins to antibodies, and we demonstrated the advantage of having a polymeric spacer between the antibody and the drug. We also showed that the drug, conjugated to simple polymers such as dextran, can be useful for drug delivery and targeting, and in a series of surprising control experiments we established that in many cases the best results could be obtained when the free drug was given as a mixture with the free antitumor antibody (Hurwitz et al. 1978). This approach was recently adopted by others and eventually led to efficient treatment of human breast cancer by recombinant humanized anti-HER2 antibody (Herceptin) in a mixture with paclitaxel and doxorubicin.

We have recently introduced a new system based on antibody-directed enzyme prodrug therapy (ADEPT), using antibody-conjugated alliinase to produce a cytotoxic agent, allicin, in situ at the site of the cancer (Miron et al. 2003). We have also applied the avidin–biotin system to target drugs to different organs. The affinity therapy is now being pursued in many other laboratories around the world and is already at the stage of clinical trials (Hurwitz et al. 2000).


   The avidin–biotin system
TOP
 Introduction
 Scientific life
 Affinity chromatography
 Affinity labeling
 Affinity therapy
 The avidin-biotin system
Finale
 References
 
The avidin–biotin system, which we started together with Ed Bayer, has become a "universal" tool in most of the fields of the biological sciences, thanks to studies we commenced in the early-1970s. The avidin–biotin system can contribute to the interaction between any two biomolecules in an indirect manner as follows: Biotin can be chemically coupled to a binder molecule (e.g., a protein, DNA, hormone, etc.) without disturbing the interaction with its target molecule; avidin can then be used to "sandwich" between the biotinylated binder and a reporter molecule or probe. This allows us to perform a variety of tasks with our system, including localization, identification, assay, etc. of the binder or target molecule. Consequently, the avidin-biotin system can emulate in many ways what had previously been accomplished using radioactive probes, which can thus be replaced by a variety of colorful alternatives. Moreover, we can now introduce many other new and often unanticipated applications (Wilchek and Bayer 1988).

The application of this system is really unlimited. The overall approach of the avidin–biotin system and a list of the various targets, binders, probes, and many of the applications are shown in Figure 2Go. In general, we have no control over the targets and binders if we want to study biological molecules, since the target is an integral part of the experimental system and we are limited to the types of interacting binders. On the other hand, we have complete control over the other parts of the system, including the coupling of biotin and the probe and/or the selection of a capture or detection system.



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  		Figure 2. Overall summary of the avidin–biotin system. The principle of the two approaches is shown. In Approach A, the direct approach, a target is labeled with a biotinylated binder, which is labeled subsequently with an avidin-probe conjugate. In Approach B, the sandwich approach, free avidin mediates the binding of a biotinylated probe to the biotinylated binder. Various target–binder pairs, representative probes, and many of the applications are listed.

 
The covalently coupling of biotin to one or more components of our system is usually done chemically, using a biotin-containing reagent that we introduced originally. Many of our biotinylating reagents are now commercially available. Thus, with comparatively little effort, we can insert a biotin molecule into a target, a binder, or a probe, with very little effect on their respective activities.

Avidin (or its bacterial cognate, streptavidin) can be introduced into an experimental system for several different purposes: for example, to capture, to detect, or to perturb. Historically, we employed these approaches in the initial development of the avidin–biotin system. The biotinylated binder and its target can be any of the molecules listed in Figure 2Go, and the system is amenable to an unlimited number of permutations and combinations, dictated only by the purpose and requirements of the experimental system and the imagination and skill of the scientist. Throughout the years, the number and nature of the applications has indeed grown, and I certainly have no space to describe them in detail, since we have already described them thoroughly in previous reviews (Wilchek and Bayer 1988).

In recent years, we have turned toward the study of the protein chemistry of the avidin–biotin complex in order to understand how such a strong binding site is being formed. These studies have culminated in the determination of the three-dimensional structure of the avidin–biotin complex by X-ray crystallography (Livnah et al. 1993). The extension of this study will hopefully enable us to design and chemically synthesize specific artificial recognition sites, a direction which may eventually prove even more far-reaching in its scope and application than that of the natural system (Domovich-Eisenberg et al. 2004).

More than two decades have elapsed since we wrote our first reviews on the avidin–biotin system. We have always been well aware of its unique properties and universal technological application. In the early days, our friends chided us, claiming that the avidin–biotin system was not very interesting or relevant. Later, the same friends told us that the system had long passed its prime and will soon be obsolete. More recently, our friends have told us that the system has always been around. I am always happy to review manuscripts in which the major claim is a superior system or alternative to the avidin–biotin system. I usually immediately approve the manuscript but most of the time there have not been sequels to the original article. On the other hand, the avidin–biotin system continues to display a tremendous level of vitality, proving indispensable for a variety of applications and generally irrepressible in its utility in a growing number of fields. Rather than dying a natural death, the system continues to develop in many wonderful and surprising directions; many scientists from fields of physics, materials science, nanotechnology, and biotechnology and other areas we never could have foreseen are now devising new and fantastic applications based on the (strept) avidin–biotin complex. There is no doubt that the avidin–biotin system will continue to thrive and will continue to surprise us in the future.


   Finale
TOP
 Introduction
 Scientific life
 Affinity chromatography
 Affinity labeling
 Affinity therapy
 The avidin-biotin system
 Finale
References
 
My life with biorecognition has really been a life of affinity, thanks to the friendship of my teachers, my coworkers, my students, my wife, and family. I particularly want to acknowledge Pedro Cuatrecasas, Ed Bayer, Talia Miron, and, of course, my wife, Ester, for their continued support and devotion throughout the years. I already apologize to all the others who have been so close to me during the years, and I will find an appropriate future opportunity to acknowledge them all.


   Footnotes
 
1 I thank the Protein Society and its committee for awarding me the Christian B. Anfinsen award—my beloved teacher—and for asking me to write a short description of my work. Since I do not know exactly for which part of my work I received the award, I decided to summarize some of my studies which I consider worthwhile. Back

Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04986004.


   References
TOP
 Introduction
 Scientific life
 Affinity chromatography
 Affinity labeling
 Affinity therapy
 The avidin-biotin system
 Finale
 References
 
Cuatrecasas, P., Wilchek, M., and Anfinsen, C.B. 1968. Selective enzyme purification by affinity chromatography. Proc. Nat. Acad. Sci. 61: 636–643.[Free Full Text]

———. 1969. Affinity labeling of the active site of Staphylococcal nuclease: Reactions with bromoacetylated substrate analogues. J. Biol. Chem. 244: 4316–4329.[Abstract/Free Full Text]

Domovich-Eisenberg, Y., Pazy, Y., Nir, O., Raboy, B., Bayer, E.A., Wilchek, M., and Livnah, O. 2004. Structural elements responsible for conversion of streptavidin to a pseudoenzyme. Proc. Nat. Acad. Sci. 101: 5916–5921.[Abstract/Free Full Text]

Edelhoch, H., Brand, L., and Wilchek, M. 1967. Fluorescence studies with tryptophyl peptides. Biochemistry 6: 547–559.[CrossRef][Medline]

Edelhoch, H., Lippoldt, R.E., and Wilchek, M. 1968. The circular dichroism of tyrosyl and tryptophanyl diketopiperazines. J. Biol. Chem. 243: 4799–4805.[Abstract/Free Full Text]

Gavish, M., Zakut, R., Wilchek, M., and Givol, D. 1978. Preparation of a semisynthetic antibody. Biochemistry 17: 1345–1351.[CrossRef][Medline]

Hurwitz, E., Levy, R., Maron, R., Wilchek, M., Arnon, R., and Sela, M. 1975. The covalent binding of daunomycin and adriamycin to antibodies, with retention of both drug and antibody activities. Cancer Res. 35: 1175–1181.[Abstract/Free Full Text]

Hurwitz, E., Maron, R., Bernstein, A., Wilchek, M., Sela, M., and Arnon, R. 1978. The effect in vivo of chemotherapeutic drug-antibody conjugates in two murine experimental tumor systems. Int. J. Cancer 21: 747–755.[Medline]

Hurwitz, E., Klapper, L.N., Wilchek, M., Yarden, Y., and Sela, M. 2000. Inhibition of tumor growth by poly(ethlene glycol) derivatives of anti-ErbB2 antibodies. Cancer Immunol. Immunother. 49: 226–234.[CrossRef][Medline]

Livnah, O., Bayer, E.A., Wilchek, M., and Sussman, J. 1993. Three-dimensional structures of avidin and the avidin-biotin complex. Proc. Nat. Acad. Sci. 90: 5076–5080.[Abstract/Free Full Text]

Miron, T., Mironchik, M., Mirelman, D., Wilchek, M., and Rabinkov, A. 2003. Inhibition of tumor growth by a novel approach: In situ allicin generation using targeted alliinase delivery. Mol. Cancer Ther. 2: 1295–1301.[Abstract/Free Full Text]

Morpurgo, M., Hofstetter, H., Bayer, E.A., and Wilchek, M. 1998. A chemical approach to illustrate the principle of signal transduction cascades using the avidin-biotin-system. J. Am. Chem. Soc. 120: 12734–12739.[CrossRef]

Strausbauch, P.H., Weinstein, Y., Wilchek, M., Shaltiel, S., and Givol, D. 1971. A homologous series of affinity labeling reagents and their use in the study of antibody binding sites. Biochemistry 10: 2631–2638.[CrossRef][Medline]

Wilchek, M. 1972. Purification of the heme peptide of cytochrome c by affinity chromatography. Anal. Biochem. 49: 572–575.[CrossRef][Medline]

———. 1984. Affinity labeling: from nuclease to cells. In The Impact of Protein Chemistry on the Biomedical Sciences (eds. A.N. Schechter et al.), pp. 91–105. Academic Press, Orlando, FL.

Wilchek, M., and Bayer, E.A. 1988. The avidin-biotin complex in bioanalytical applications. Anal. Biochem. 171: 1–32.[CrossRef][Medline]

Wilchek, M., Bocchini, V., Becker, M., and Givol, D. 1971a. A general method for the specific isolation of peptides containing modified residues, using insoluble antibody columns. Biochemistry 10: 2828–2834.[CrossRef][Medline]

Wilchek, M., Salomon, Y., Lowe, M., and Selinger, Z. 1971b. Conversion of protein kinase to a cyclic AMP independent form by affinity chromatography on N0-caproyl 3',5'-cyclic adenosine monophosphate-Sepharose. Biochem. Biophys. Res. Commun. 45: 1177–1184.[CrossRef][Medline]

Wilchek, M., Miron, T., and Kohn, J. 1984. Affinity chromatography. Meth. Enzymol. 104: 3–56.[Medline]

Zioudrou, C., Wilchek, M., and Patchornik, A. 1965. Conversion of the L-serine residue to an L-cysteine residue in peptides. Biochemistry 4: 1811–1822.[CrossRef]


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