MODIFIED AVIDIN AND STREPTAVIDIN MOLECULES AND USE THEREOF

Abstract: Biotin-binding modified avidin-type molecules are provided in which the essential tyrosine residue in the bioting-binding site is modified in such a way that its pKa is decreased in comparison to the pKa of the unmodified tyrosine residue in the corresponding unmodified avidin-type molecule. The avidin-type molecules include: (i) native egg-white avidin; (ii) recombinant avidin; (iii) deglycosylated forms of avidin; (iv) bacterial streptavidin; (v) recombinant streptavidin; (vi) truncated streptavidin; and (vii) derivatives of (i)-(vi) which are modified at sites other than the essential tyrosine residue. The modification is achieved by substitution at one or both ortho positions to the hydroxy radical of the tyrosine residue by radicals such as nitro, halogen, azo and amino. The modified avidin-type molecules can be used in all applications of the avidin-biotin technology.

Description: MODIFIED AVIDIN AND STREPTAVIDIN MOLECULES AND USE THEREOF Field and Background of the Invention The present invention relates to an avidin-type molecule which is modified at the binding-site tyrosine residue, known to be critical to the binding of biotin. The modified avidin is still capable of binding biotin or a biotinylated ligand under specific conditions, but upon altering these conditions, for example, high pH or competition with biotin, the bound biotin moiety or biotinylated ligand is removed. The invention thus provides a reversible form of avidin for use in avidin-biotin technology, thus "correcting" one of the major disadvantages of the avidin molecule for various applicative purposes, i.e., the extreme denaturing conditions required to disrupt the avidin-biotin complex. These drastic conditions necessary to dissociate the avidin-biotin complex usually inactivate irreversibly the biological activity of the biotinylated component, thus rendering it unsuitable for subsequent use. Avidin (from egg-white) and streptavidin (from Streptomyces avidiniϊ) are two related proteins that bind biotin with similar dissociation constants of about 10 * M (Green, 1975). In addition to the binding of biotin, many of their physical properties are quite similar. Both, for example, are constructed of four non-covalently attached identical subunits, each of which bears a single biotin-binding site. The subunit M- values are also very similar. Moreover, several short stretches in the sequences of the two proteins are preserved, particularly two Trp-Lys stretches that occur at approximately similar positions (Argarana ei al., 1986). We have previously shown (Gitlin et al, 1987, 1988α) that certain lysine and tryptophan residues are involved in the biotin binding in both proteins (Gitlin et al, 1988b). More recently, it was shown that both avidin and streptavidin exhibit the same three dimensional fold, and that most of the binding site residues are identical or similar (Weber et al, 1989). The binding site geometry and bonds formed between both proteins with the biotin molecule are indeed very similar. Despite these similarities, several differences exist between the two proteins. Avidin is a disulphide-bridged glycoprotein containing two methionine residues, whereas streptavidin is not glycosylated and is devoid of sulphur-containing amino acid side chains. Another significant difference is in the tyrosine content. Avidin has only one tyrosine residue (Tyr-33), whereas streptavidin has six tyrosine residues at positions 22, 43, 54, 60, 83 and 96. Interestingly, the single tyrosine residue of avidin is located in a region which contains a sequence identical with that of one of the streptavidin tyrosine residues (Tyr-43 in the stretch Thr-Gly-Thr-Tyr). This tyrosine residue occupies a prominent position in the biotin-binding site and the chemical modification of the tyrosine hydroxyl group leads to irreversible inactivation of the avidin molecule (Gitlin et al, 1990) Each avidin monomer binds one molecule of biotin. The unique feature of this binding, of course, is the strength and specificity of formation of the avidin-biotin complex. The resultant affinity constant, estimated at 1.6 x 10 15 M 1 for avidin and 2.5 x 10 M 1 for streptavidin (Green, 1990), is the highest known for a protein and an organic ligand. It is so strong that biotin cannot be released from the binding site, even when subjected to a variety of drastic conditions such as high concentrations of denaturing agents at room temperature, e.g., 6 M guanidinium hydrochloride, 3 M guanidinium thiocyanate, 8 M urea, 10% β- mercaptoethanol or 10% sodium dodecyl sulfate. Under combined treatment with guanidinium hydrochloride at low pH (1.5) or upon heating (>70°C) in the presence of denaturing agents or detergents, the protein is denatured, and biotin is dislodged from the disrupted binding site. Avidin recognizes biotin mainly at the ureido (urea-like) ring of the molecule. The interaction between the binding site of avidin with the sulfur-containing ring of the valeric acid side chain of the vitamin is of much lower strength. The relatively weak interaction between the carboxy-containing side chain of biotin and avidin means that the former can be modified chemically and attached to a wide variety of biologically active material; the biotin moiety of the resultant derivative or conjugate is still available for interaction with avidin. In turn, the avidin can be derivatized with many other molecules, notably "probes" or reporter groups of different types. This is the crux of avidin-biotin technology (Wilchek and Bayer, 1990). Thus, a biologically active target molecule in an experimental system can be "labeled" with its biotinylated counterpart (a binder), and the product can then be subjected to interaction with avidin, either derivatized or conjugated with an appropriate probe. The use of the egg-white avidin in the avidin-biotin technology is sometimes restricted due to the high basicity (pl~10.5) and presence of sugar moieties on the avidin molecule, which may lead to nonspecific or otherwise undesired reactionsJn recent years, the bacterial protein, streptavidin, has largely replaced egg-white avidin for most applications in avidin-biotin technology. However, the problems with streptavidin (high cost and biotin-independent cell binding) have prompted renewed interest in egg-white avidin as the standard for avidin-biotin technology. For this purpose, modified avidins exhibiting improved molecular characteristics both over the native protein (and previous derivatives thereof) as well as over streptavidin, have been prepared, such as N-acyl avidins, e.g., N- formyl, N-acetyl and N-succinyl avidins. These derivatives of avidin reduce the charge of the protein, but they are all prepared via covalent attachment to the available lysines of avidin, and the consequent blocking of the free amino groups hinders subsequent preparation of other types of conjugates (notably protein-protein conjugates such as avidin-labeled enzymes) which are often prepared by crosslinking via lysine residues using bifunctional reagents (e.g., glutaraldehyde). A more useful and effective alternative to lysine modification is the modification via arginines. In this case, the pi of the protein is efficiently reduced and the lysines are still available for subsequent interaction. Two different derivatives of avidin which are modified in this manner are commercially available. One, ExtrAvidin , can be obtained in various functionally derivatized or conjugated forms from Sigma Chemical Company (St. Louis, MO). A second , NeutraLite Avidin™ (a product of Belovo Chemicals, Bastogne, Belgium) is additionally modified and can be purchased in bulk quantities. Although the reduction of the pi of egg-white avidin solves one of the problems, the presence of the oligosaccharide residue remains a serious source of nonspecific (biotin- independent) interaction which restricts its application. The return of egg-white avidin as the standard for avidin-biotin technology has been contingent upon the removal of its sugars. The possibilities for removing a sugar from a glycoprotein are quite limited; it is possible to do so either chemically or enzymaticaUy. The chemical methods currently available, e.g., using HF or periodate oxidation, are either destructive or inefficient. The well known enzymatic method, which employs N-glycanase (Tarentino et al, 1984), is usually very expensive and not very effective for avidin when conventional methodology is used. Eventually, a viable procedure for deglycosylation was established and the resultant product was subsequently modified chemically via the arginines and is known under the trade mark NeutraLite Avidin™ (Belovo Chemicals). In spite of all these improvements, one of the main problems in the several applications of the avidin-biotin technology is the lack of reversibility of the binding and the difficulty of separating the avidin and the biotin moieties at the end of the process, without denaturation of the avidin or damaging or inactivating the biological material which had been attached via the biotin bridge. Alternatively, it would be advantageous (particularly for industrial use) to remove damaged or inactivated material from an avidin column, thus reconstituting the column for attachment of a new sample of biotinylated component. It is an object of the present invention to provide modified avidins which are still biotin-binding and can be advantageously used in methods employing the avidin-biotin technology in which reversibility of the method is desired or is an advantage. Nitration of tyrosine residues in model peptides and proteins using tetranitromethane has been described (Riordan et al, 1966; Sokolovsky et al, 1967) In a previous work of the present inventors (Gitlin et al, 1989), a nitrotyrosine derivative of avidin was prepared by nitration of egg-white avidin dissolved in 9M-urea with tetranitromethane (TNM) The resultant nitro-avidin preparation was inactive, i.e it failed to bind biotin, because the nitration was carried out on a denatured form of avidin (in the presence of urea) The nitro- avidin thus prepared is entirely inadequate for use in avidin-biotin technology. I-labelled avidin and streptavidin have been prepared for analytical purposes The single tyrosine residue of each avidin subunit is not readily accessible to iodination. Avidin is rendered susceptible to iodination (chloramine T method) by the introduction of 3-(p- hydroxyphenyl)propionyl groups and thus 125 I-labelled avidin containing said groups was prepared 125 I-labelled Bolton-Hunter reagent can also be employed to label avidin (Finn and Hofmann, 1985) 125 I-streptavidin was produced by iodination of streptavidin with Na I using the iodogen method (Suter et al, 1988). Unlabelled iodinated avidin and streptavidin have not been described heretofore. Azotization and amination of tyrosine residues in model peptides and proteins, e g ribonuclease A, has been previously described (Gorecki et al, 1971; Sokolovsky et al, 1967) Summary of the Invention The present invention relates to a biot -binding modified avidm-type molecule selected from the group of molecules comprising (1) native egg-white avidin, (u) recombinant avidin, (m) deglycosylated forms of avidin, (iv) bacterial streptavidin, (v) recombmant streptavidin, (vi) truncated streptavidin, and (vn) derivatives of (ι)-(vι) which are modified at sites other than the essential tyrosine residue, cnaracteπzed in that in said biotin-bindmg modified avidm-type molecule the essential tyrosine residue in the biotin- binding site is modified in such a way that its pKa is decreased in compaπson to the pKa of the unmodified tvrosme residue in the corresponding unmodified avidm-type molecule The modified avidin-type molecule of the invention has one or more electrophilic and/or nucleophihc groups on the essential tyrosine residue of the avidm-tv pe molecule, and mav be exemplified by compounds wherein the modified tyrosine is of the formula wherem X* and X 2 are each a radical selected from nitro, halogen, NR*R 2 and ~ = R 3 in wnich R* and R 2 are each selected from hydrogen, C r C 8 alkyl and C C 6 carboxvπc acyl, and R 3 is aryl substituted by an acidic radical In one preferred embodiment of the invention, the avidm-type molecule is modified by addition of one or more electrophilic groups on the tyrosine residue For example, avidm or streptavidin mav be modified by nitration or halogenation, preferably iodination, of the tyrosme residue, as depicted m Fig 1 (X* or X 2 is N0 2 or halogen, preferably I), thus decreasmg the pKa of the tyrosme residue in the biotin-binding site from 10 5-12 5 to 6 5- 8 5, preferably to about 7 0 Such modification of the tyrosme residue in the avidm-type molecule involves the addition of one or more electrophilic group(s) at the ortho posιtιon(s) (adjacent to the hvdroxvl grouD) on the tyrosine ring As an example of this type of modified avidm, the nitration of the tvrosme residue using tetranitromethane (TNM) was employed The resultant nitrotvrosine-contaimng avidin and streptavidin, hereinafter referred to as "nitro-aviαm" and niro-iireotaviGtn ', respectively, have been studied extensiv ely in order to define relatives mild conditions for releasing the biotin moiety or the biotinylated ligand, e g , a biotinylated antibody, enzyme, nucleic acid or cell In the present invention, the nitration of avidin was performed under nondenatuπng conditions, and the resultant nitro-avidin and nitro-streptavidin were shown to bind a biotinylated ligand efficiently and tightly at pH 4 When the pH was elevated to 8, the biotinylated ligand was still retained on the column However, when the pH was elevated to 10, the biotinylated ligand was released Alternatively, at lower pH (e g , between the range of pH 4 to 8), the biotinylated ligand could be released by exchange usmg free biotm These characteristics of the nitro-avidm and of the nitro-streptavidin provide forms of avidm and streptavidin which are appropriate for a variety of applications These materials have been used according to the present invention for the binding and subsequent release of several examples of biotinylated ligands to nitro-avidin and nitro-streptavidm immobilized onto a Sepharose resin as well as for the attachment and release of biotinylated gands to mtro- avidin and nitro-streptavidin adsorbed to microtiter plates In another embodiment the present invention relates to halogenated, more preferablv to unlabelled iodinated avidin or streptavidin Iodination of the tyrosine residue in avidm or streptavidin with KI using the chloramine T procedure, yielded mono- and/or dnodotyrosme- avidin or -streptavidin, shown also to be effective reversible forms of avidm A further embodiment of the invention regards avidin-type molecules modified at the essential tyrosme residue by one or more more nucleophi c groups selected from NR-R-, and -N=NR3 In the azo derivatives of the mvention, l e the compounds wherem X, and/or X 2 is — N=NR 3 , R 3 is aryl, preferably carbocyclic aryl, most preferably, phenyl, substituted by an acidic radical selected from carboxyl and a residue of an inorganic acid such as phosphoric, arsonic or sulfonic acid The azo derivatives according to the mvention are prepared from the corresponding p-ammo derivatives, e g p-arsaniiic acid, anthram c acid, p-ammooenzoic acid, sulfam c acid and p-ammophosphoπc acid, by diazotization with NaN0 and reaction of the resultmg diazomum salt with the avidm-type moecule of choice In the ammo derivatives of he invention, 1 e the compounds wherem X, and/or X 2 is NR, R 2 R, ana R 2 are eacn selected from H, alkyl and acyl Tne alkvl radical is preferably a C -C 3 straight or oranched alkyi examples being methv l ethvl propv l isopropyl, butvi, hexyl, octyl. The acyl radical is preferably a C C 6 carboxylic acyl such as acetyl, propionyl, butiryl, succinyl, or benzyloxycarbonyl. The amino derivatives of the invention may be prepared by reduction of the corresponding azo derivatives with sodium hydrosulfite Na 2 S s O 4 or by reduction of the corresponding nitro derivatives with Na 2 S s O 4 and, if desired, the amino group is further alkylated or acylated by standard methods The modified avidin-type molecules of the present invention can be used in the very many applications of the avidin-biotin technology. Brief Description of the Drawings Fig 1 shows a reaction scheme for preparation of a tyrosine-modified avidin-type molecule of the invention and its use in a reversible method using avidin-biotin technology. Fig. 2 shows levels (%) of nitration of avidin as a function of tetranitromethane (TNM) concentration (mM). as described in Example l(i) Fig 3 shows the effect of pH on the binding of biotinylated bovine serum albumine (BSA) to a Sepharose-nitro-avidin resin, as described in Example 2(i) Fig 4 shows the effect of pH on the binding of biotinylated alkaline phosphatase to microtiter plates containing adsorbed nitro-avidin, as described in Example 2(ii) Fig 5 shows the comparison between biotin-binding activity of avidin (closed circles) and nitro-avidin (open circles), as described in Example 2(iii) Fig. 6 shows pH-induced release of biotinylated BSA from a Sepharose-nitro-avidin resin, as described in Example 3(i) Fig 7 shows pH-induced release of biotinylated BSA from a Sepharose-nitro- streptavidin resin, as described in Example 3(ii) Fig 8 shows release of biotinylated BSA from a Sepharose-nitro-avidin resin by competition with free biotin. as described in Example 3 (iii) Fig 9 shows biotin-induced elution of biotinylated BSA as a function of pH, as described in Example 3(iv) The free biotin was dissolved in buffers of different pH, ranging from 4 to 10 Elution at pH 10 in the absence of biotin is shown for comparison. Fig 10 shows the release of biotin from biotin-blocked sites of nitro-avidin, as described in Example 4 Figs. 11 A-B show the results of repeated application and elution of biotinylated BSA from a nitro-avidin-Sepharose column, as described in Example 5. In Fig. 1 1 A, identical samples of biotinylated BSA were applied successively (at pH 4 using Buffer A) to, and eluted (at pH 10 using Buffer C) from, a column containing a Sepharose-nitro-avidin resin. Fig. l 1 B shows that accumulation of the eluted fractions gave essentially identical levels of protein bound and eluted per cycle. Fig. 12 shows the release of biotinylated protein A from a column containing a Sepharose-nitro-avidin resin following purification of immunoglobulin from whole rabbit serum, as described in Example 6. Fig. 13 shows the SDS-PAGE profile of samples from the column in Fig. 12: Lane A, whole rabbit serum (applied fraction); Lane B, column effluent; Lane C, peak eluted by Buffer A, pH 4; Lane D, peak eluted by Buffer C, pH 10; Lane E, commercial preparation of rabbit immunoglobulin; Lane F, biotinylated protein A standard. Fig 14 shows the effect of pH on the releasing of biotinylated horseradish peroxidase from microtiter plates containing adsorbed iodinated avidin, as described in Example 7. Detailed Description of the Invention The term "avidin-type molecule" as used herein refers to the native egg-white glycoprotein avidin, to deglycosylated forms of avidin, to bacterial streptavidms produced by selected strains of StreDtomyces, e.g., Streptomyces σvidinii, to truncated streptavidins, and to recombinant avidin and streptavidin as well as to derivatives of native, deglycosylated and recombinant avidin and of native, recombinant and truncated streptavidin, which are modified at sites other than the essential tyrosine, for example, N-acyl avidins, e.g., N- acetyl, N-phthaiyl and N-succinyl avidin, and the commercial products ExtrAvidin™ and Neutralite Avidin™. All forms of avidin-type molecules are encompassed by the present invention, both native and recombinant avidin and streptavidin as well as derivatized molecules, e.g. nongiycosyiated avidins, N-acyl avidins and truncated streptavidins. Some of these materials are commercially available, e.g. native avidin and streptavidin, nonglycosylated avidins, N- acyl avidins and truncated streptavidin, or can be prepared by well-known methods (see Green, 1990, for preparation of avidin and streptavidin: Hiiler et al, 1990, for preparation of non-glycosylated avidin; Bayer et al, 1990, for the preparation of streptavidin and truncated streptavidin) Recombinant avidin and streptavidin can be prepared by standard recombinant DNA techniques, for example, as described by Chandra and Gray, 1990, for recombinant avidin, and by Argarana et al , 1986, for recombmant streptavidin The "biotinylated ligands" that can be used with the modified avidins of the invention in methods of application of the avidin-biotin technology, are biotinylated forms of desired ligands such as proteins, e g antibodies, enzymes, lectins, or carbohydrates and glyco¬ conjugates, e g glycoproteins, ganghosides, heparin, polysaccharides, or nucleic acids, l e DNA and RNA, or phages, viruses, bacteria and other cells, wherein said ligands are covalently linked to biotin or to a homolog, analog or derivative thereof Many biotinylated ligands are commercially available or can be prepared by standard methods ( see, for example, Bayer and Wilchek, 1992a) The modified avidin-type molecules of the invention, mainly avidin and streptavidin derivatives modified at one or both ortho positions of the binding-site tyrosine residue (see Fig 1), are suitable for reversible interaction with the biotin moiety, and thus constitute important new tools for avidin-biotin technology These modified avidin-type molecules allow to remove, under mild conditions, free biotin and/or biotinylated ligands, e g biotinylated enzymes and other biotinylated biologically active materials, from immobilized ortho-phenol-modified avidin-type molecules or from soluble complexes which comprise the modified avidin together with the biotinvlated ligand in solution Such mild conditions may consist of excess concentrations of biotin, high pH, e g pH 10, relatively low nondenatuπng concentrations of urea, guanidine or thiocyanate, heat and/or combinations thereof In one preferred embodiment, removal of the biotin or the biotinylated moiety from the immobilized modified avidin is carried out by change of the pH, for example raising the pH to 10 In another preferred embodiment, removal is carried out by adding an excess of biotin, for example, a solution of 0 6 mM biotin is passed through the modified avidin column to displace the biotinylated component The modified avidin-type molecules of the invention are suitable for use in any method employing the avidin-biotin technology, particularly in those methods wherein reversibility of the avidin-biotin binding is an advantage, for example, as reversible immobilization columns for use in affinity chromatography for removal of affinity ligands, to remo e immobilized enzvmes thus creating a reversible enzvme reactor to disrupt soluble biological complexes in solution consisting of biotinylated material crosslinked via an avidm bridge, to produce high-afinity phage libraries, and for use in cell separation, thus facilitating the release of a viable or intact cell together with the recognition component or ligand, e g antibody, from a resin, or to counteract agglutination of such cells by dissociating the avidin bridge The invention also relates to a modified avidm-type molecule of the invention attached to a solid support or matrix Any solid support used in the art is suitable such as, but not limited to, resins, microtiter plates, glass beads, magnetic beads and the like The attachment of the avidin to the solid support may be covalent or noncovalent and is carried out by standard methods In one preferred embodiment, the modified avidin-type molecule is immobilized onto a resin, preferably Sepharose, and the thus obtained Sepharose-nitro-avidin affinity resm may be poured into a column for isolation procedures (Bayer and Wilchek, 1992b) In the description herein the term "avidin-Sepharose column" will be used for a column that contains a modified avidin-type molecule of the invention immobilized onto a Sepharose resin These columns are useful particularly for separation procedures In another embodiment of the invention, the modified avidin-type molecule is attached to wells of microtiter plates In the well-known affinity chromatography procedure, which is the prototype of all affinity methodologies, a binding hgand,e g , an antibody or receptor, is attached to a solid support, such as Sepharose This can be accomplished by biotinylation of the said ligand, and subsequent immobilization to Sepharose via an avidin or streptavidin bridge The resultant avidin-Sepharose column is then used as a handle to isolate and purify mateπal which interacts with said biotinylated ligand In many cases, it would be advantageous to separate the biotinylated ligand from the avidin- Sepharose column, either to recover the ligand itself, which might be precious or delicate in nature, or to reconstitute the column for alternative usage This can be accomplished by using a column which contains a modified avidin-type molecule of the present invention to immobilize the biotinylated ligand, which can eventually be released from the column by addition of either alkaline solutions (e g , Buffer C, pH 10) or by adding excess biotin (e g , 0 6 mM at any pH) Either a new type of biotinylated ligand or a fresh batch of the same biotinylated ligand can then be added to the reconstituted avidin column The avidin-biotin system has been used to separate cells by a vaπetv of methods One approach is to use a biotinylated ligand (e g , antibody) which recognizes a cell surface molecule (e g , surface antigen) The biotinylated ligand can be bound to a column or other type of matrix, such as magnetic beads, via an avidm or streptavidin bridge Alternatively, a suspension of a mixed cell population ca be treated with the biotinylated ligand and the cells bearing the interacting surface molecule can be agglutinated using avidin in solution Commonly, this approach has been used simply to selectively ' remove" a given population of cells from the mixed population Once bound to the matπx or agglutinated by avidin, the affinity interactions involved (I e , between the biotinylated ligand and surface molecule, and between the avidm and biotin) cannot be easily disrupted, in a manner which would preserve cell integrity or viability For example, disruption of the interaction between an antibody and an antigen usually requires conditions, such as low pH, which are damaging to most cells The non-interacting cells, however can be recovered In order to recover the bound cell population, a column containing a modified avidin-type molecule of the present invention can be used and a biotm-contammg solution (e g , 0 6 mM of biotin under isotonic conditions, e g , 0 15 M NaCl at pH 7, or such excess concentrations of biotm in a suitable tissue- culture medium) can be used to release the cells (together with the biotinylated ligand) from the column or the agglutinated cells can be dispersed using the same biotm-containmg solution Immobilized enzymes are largely used in the food, pharmaceutical and chemical industries (Katchalsky-Katzir, 1993) One of the problems with immobilized enzymes for use in enzyme reactor systems is that the enzyme or its matπx undergoes a type of agmg process For example, enzymes are notoriously sensitive proteins, they often have a definitive half-life, and, in time, they may be inactivated either during use or upon storage For industrial usage, it would thus be advantageous to reuse the immobilizing matrix once the bound enzyme has become useless An enzyme reactor consistmg of a biotinylated enzyme bound to a column containmg a modified avidm-type molecule according to the present invention can thus be reconstituted by removing the inactivated biotinylated enzyme by addition of either alkaline solutions (e g , Buffer C, pH 10) or by addmg excess biotm (e g , 0 6 mM at any pH") A fresh batch of biotinylated enzvme can then be added to the reconstituted modified avidin column Similarly, cells can be immobilized to a modified avidm column and used as a cellbaseu reactor system The cells can be released using a biotiπ-containing solution (e g , 0 6 m of biotm uncer isotonic conditions e g , 0 15 M NaC. at pH 7, or sucn excess concentrations of biotin in a suitable tissue-culture medium), and the modified avidin column can be thus reconstituted. In the phage library technique, a binding ligand (e.g., antibody, receptor) is attached to a solid support such as a microtiter plate. This is often accomplished by biotinylation of the said ligand, and subsequent immobilization to the plate via a streptavidin bridge. Filamentous bacteriophages (e.g., M13) are then added, and those phages, which contain surface peptides that are capable of interacting with the immobilized ligand, thus bind to the plate. Unspecifically bound phages are removed by washing with low concentrations of neutral detergents such as Tween 20. The bound phages are subsequently released from the plate, usually by reducing the pH which disrupts the interaction between the immobilized ligand and the surface peptides on the phage. One of the potential problems in this approach is that some phages may still be bound to the plate via very high affinity interactions. These phages would be of interest specifically due to their high affinity peptides to the biotinylated ligand. Thus, by using the modified avidin or streptavidin of the present invention, the high affinity phages can be released from the microtiter-plates together with the biotinylated ligand, by addition of either alkaline solutions (e.g., Buffer C, pH 10) or by adding excess biotin (e.g., 0.6 mM at any pH). The recovered high-affinity peptides bound to the phage can then be enriched by subsequent infection of bacteria, and by established phage library procedures (see, for example, Scott, 1992). Gene enrichment and DNA isolation can be achieved by complexing biotinylated DNA with a modified avidin-type molecule or a modified avidin column according to the present invention by known methods (see Wilchek and Bayer, 1988). In the past, proteolytic enzymes, such as proteinase K, have been used to digest the avidin in order to free the biotinylated DNA from the complex. Using the modified avidin-type molecule of the present invention, the biotinylated DNA can be released using alkaline solutions (e.g., Buffer C, pH 10) or by adding excess biotin (e.g., 0.6 mM at any pH). Biosensors consist of a biological-sensing element which confers specificity and a transduction function (i.e., electrochemical, optical, calorimetric or acoustic) which convers a biological event into a response that can be further processed and quantified. The biological ligand can either be catalytic (e.g., enzymes, bacterial cells, tissues) or noncatalytic (e.g.. antibodies, receptors, DNA). Such detectors rely on the immobilization of one of the interacting components onto the sensing surface, and the resultant constituents of the completed biosensing apparams are notably expensive The avidm-biotin system has been used in the past as a general method for immobilizing such ligands onto biosensors The capacity to replace the native avidm or streptavidin with the modified avidins according to the present invention, would provide a reversible type of biosensor which would be an advantage in cost and convenience Thus, the biotinylated ligand can be immobilized to the biosensor via the modified avidm or streptavidin and when desired the biotunvlated ligand can be released using alkaline solutions (e g , Buffer C, pH 10) or by adding excess biotin (e g , 0 6 mM of biotm at any desired pH, ionic strength conditions, etc ) The modified avidin-type molecule of the biosensor can then be charged with either the same or a different biotinylated gand The present mvention also provides a process for the recovery of either the avidin- column cr the biotinylated ligand in a method employing the aviαin-biotin technology, which comprises (1) immobilizing a biotinylated ligand onto a column containing a modified avidin-type molecule of the invention attached to a resm, (n) carrying out a desired reaction or separation process with the thus immobilized biotinvlated ligand, (in) removing the biotmv.atec ligand from the immobilized modified avidin column by altering the conditions, and (iv) recovermg the biotinylated ligand and/or the modified avidm-column for further use Tne biotinylated ligand may be removed from the immobilized modified aviαin-column by raising the pH, heating, adding excess concentrations of biotm, or low concentrations of urea, guanidine or thiocyanate and/or combmations thereof In preferred embodiments, the biotinylated ligand is removed from the immobilized modified avidm-column by raising the pH to 10, or by addmg 0 6 mM biotin The mvention will now be illustrated by the followmg non- miting examples EXAMPLES Materials and Methods (i) Materials. Egg-white avidm was provided by STC labs (Winnipeg, Canada) or from Belovo Chemicals (Bastogne, Belgium) Streptavidin was purified from culture filtrates of Streptomvces avidtnii using an improved immobiotm-Sepharose column as described previously (Baver et al 1990) Sepharose 4B CL was from Pharmacia (Uppsala Sweden) Tetr m τ romethane was from Fiuka Protein A and bovine serum albumin (BSA ) w ere from Sigma Chemical Co (St Louis MO, USA) (ii) Buffers Buffer A 50 mM citrate-phosphate, pH 3-6, Buffer B 50 mM Tns-HCl, pH 7-9, and Buffer C 50 mM sodium carbonate-HCl , pH 10 (iii) Biotinylation Procedures. The protems and enzymes used in the Examples were biotinylated by conventional biotmylating methods using biotmyl N-hydroxysuccinimide ester (BNHS) as described previously (Bayer and Wilchek, 1990) (iv) Immobilization of avidin and streptavidin to Sepharose was earned out by the cyanogen bromide procedure as described previously (Kohn and Wilcnek. 1984) (v) Enzyme assays. (a) Horseradish peroxidase activity Peroxidase activity was determined using 2, 2' azmo-bis (3-ethylbenz-thιazolme-6-sulfonιc acid) (.ABTS) as substrate Substrate solution included 2 5 mg of the substrate per 10 ml in Buffer A, pH 5, to whicn 10 ul of 30% hydrogen peroxide was added Color formation was measured at 420 nm (b) Alkaline phosphatase activity was determined using p-nitrophenyl phosphate as substrate Substrate solution included 10 mg of he substrate dissolved in 10 ml of 1 M diethanolamine buffer (pH 9 S) containmg 0 5 mM MgCL Color formation was measured at -UO nm (vi) Protein. Protein was determined by the Bradford method using either av idin or streptavidin (where appropriate) or BSA as a standard EXAIVEPLE 1. Preparation of nitro-avidin. nitro-streptavidin and their immobilization to Sepharose (i) Preparation of nitro-avidin Samples (5 mg in 1 ml of 50 mM Tπs-buffer, at pH 8 or above) of egg-white avidin were treated with different concentrations of tetranitromethane (TNM) (0 5-5 μl correspondmg to about 5-50 mM), for 30 mm at 23°C The samples were dialyzed overnight, once against 1 M NaCl and twice agamst double distilled water The amount of modified tyrosine in the sample was determined by ammo acid analysis As shown in Fig 2, under the conditions of the modification procedure, maximum levels (ca 70%) of modification were achieved usmg greater than 20 mM concentrations of reagent, l e , an average of about three out of the four subumts of the avidin tetramer appeared to be modified In the following experiments, the nitro-avidin used was prepared employmg 2uι (ii) Preparation of nitro-streotavidin Streptavidin ( 2 5 mg per 1 ml buffer) was suciected to nitration using higner levels of tetranitromethane 6ul corres p onding to about 50 mM), owing to the greater number of tyrosine residues per subunit of the streptavidin molecule (iii) Preparation of nitro-avidin and nitro-streptavidm immobilized to Sepharose (a) Cvanogen bromide activation of Sepharose 4B CL 40g of drained Sepharose -^B CL were washed first with water, then with 30% acetone (v/v) and finally with 60% acetone (v/v) The resm was resuspended in 6 ml of 60% acetone and cooled to 0-4°C While stirring with a magnetic stirrer, 6 ml of CNBr solution (10 g/l 00 ml acetone) were added, followed by dropwise addition of an identical volume of triethylamine (TEA) solution (15 2 g/l 00 ml acetone) for over a peπod of 1 -2 mm The activated resin was filtered and washed with 0 IM sodium bicarbonate (b) Immobilization to Sepharose Coupling of nitro-avidin and nitro- streptavidin to the activated Sepharose &B CL was performed -n 0 IM bicarbonate solution for 16h at 4°C (iv) Nitration of avidin-Sepharose and streptav ioin-Sepnarose resins A sample of 4 ml of avidm-Sepharose resm f l Λ mg avidm/ml Sepharose) prepared as described in (πι))(b) above but using unmodified avidin, was washed bv 50 mλl Tπs-buffer pH 8 and treated with 6 ul of TNM for 50 mm at 23°C The mtro-modified resm was washed first with 1 M NaCl. then with double distilled water and finally with PBS EXAMPLE 2. Binding of biotinylated proteins to nitro-avidin (i) The binding of biotinylated protems to mtro-avidin was tested in several ways In one experiment, nitro-avidm was immobilized to Sepharose by the cyanogen bromide procedure accordmg to Example l(m) using about 0 5 mg avidin per ml Sepnarose, and samples of biotinylated BSA in Buffer A, B or C (lOOul) were applied to lOOμl of the rutro- avidin Sepharose resin The effluent fractions were measured for protein The percentage of binding at different pH values was determined by subtracting the amount of protem in the effluent fractions from that applied to the resm As shown in Fig 3, optimal binding occurred at pH - 1 At higher PH (between 5 and 8), plateau levels of binding were observed Above pH 8, the binding dropped markedly and at pH 10, the binding was negligible (ii) Similar results were achieved usmg the biotinylated alkaline phosphatase enzyme in a microtiter plate assay iFig -) In this experiment nitro-aviαin pre p ared according to Example l ^i) was adsorbed * o microtiter plates (1 ug nitro-avicin. 100 ul pnospnate-Dufferec saline (PBS)/weil), the plates were blocked by a solution of 1% BSA, and samples of biotinylated alkaline pnosphatase were applied in Buffers A, B and C of different pH (37 ug/0 1 ml buffer/well) The plates were washed and the bound enzymatic fraction was determined colonmetrically by enzyme assay as described in method v(b) above using p- nitrophenyl phosphate as a substrate (iii) Using a similar microtiter plate enzyme assay procedure, the binding activity of mtro-avidm was compared with that of native (unmodified) avidm Microtiter plates, coated with avidm or mtro-avidm (1 ug/lOO ul PBS/well), were loaned with different concentrations (between 10 ng and 1 μg in 150 ul of Buffer A) of biotinylated horseradish peroxidase at the experimentally determined, optimal pH for binding (l e , pH 4) The plates were wasned and the peroxidase enzymatic activity was determined as described in method v(a) above using .ABTS as a substrate As shown in Fig 5, under these conditions, the biotm- binding performance of the unmodified avidm and of nitro-avidin were indistinguishable EXAMPLE 3. Release of biotinylated proteins from nitro-avidin (i) In order to determine the prefeπed conditions for release of biotm from the mtro- avid column, biounvlated BSA (1 5 mg/l 50 ul Buffer A. pH 4) was applied to a column containmg a nitro-av idm-Sepharose resm according to Example l(ιv) The bound material was washed with Buffers A, B and C of increasing pH, and the protem concentration of the effluent fractions was monitored As seen in Fig 6, alkaline solutions (Buffer C, pH 10) were required to release the biotinylated protein from the resm (ii) Using the same procedure, but washmg only with Buffer C, pH 10, similar results (Fig 7) were ootained using a nitro-streptavidin column prepared by coupling mtro- streptavidm to Sepharose according to Example l(iii) (iii) In a similar experiment, competition with free biotm was explored as a means to release biotmviated BSA from a column contam g a nitro-avidm-Sepharose resm prepared accordmg to Example l(iv) Biotinylated Bsa (1 5 mg/150 ul Buffer A, pH 4) was applied to a 2-ml mtro-avidm-Sepharose column A solution contaimng 0 6 mM biotm in Buffer A, pH , was passed rougn the column, and the protein concentration was momtored .As shown in Fig 8, the ma j ority of biotmyl-BSA could be released using 0 6 mM of biotm m Buffer A at pH J* The biotm- nαucεd elution of biotinvl-BSA was studied as a function of pH usmg buffers A B or C frcm pH -i to 10 (iv) A similar experiment was carried out under the same conditions with the nitro- avidin- Sepharose resin prepared according to Example l(iv), except that the free biotin was dissolved in buffers of different pH ranging from 4 to 10. As shown in Fig. 9, free biotin was an effective eluent over the entire pH range tested. Elution at pH 10 in the absence of biotin is shown for comparison. EXAMPLE 4. Blocking of unmodified biotin-binding sites Since only partial modification of the binding-site tyrosine could be achieved under the described conditions, unmodified sites could potentially pose a problem in subsequent applications of the nitro-avidin. Thus nitro-avidin samples Prepared according to Example l(i) containing different levels of nitro-tyrosine (see Fig. 2) were coated onto wells of microtiter plates (lμg/lOOμlPBS/well). Native egg-white avidin in similar concentration was used as a control. The wells were blocked with 1% BSA, and the adsorbed protein samples were selectively blocked with excess levels of free biotin using 0.6 mM of biotin in Buffer A. pH 4. The biotin which occupied the modified binding sites could be released using alkaline solutions as described above in Example 3 (Buffer C, pH 10). After blocking and alkaline treatment, the biotin-binding capacity of the partially nitrated avidin samples was determined by enzyme assay in microtiter plates using the biotinylated peroxidase system (method v(a) above). As shown in Fig. 10, the binding was found to be proportional to the extent of modification, with a maximum at about 60% modification. EXAMPLE 5. Repeated use of nitro-avidin column The stability of the column containing a Sepharose-nitro-avidin resin according to Example l (iii) was tested by repeated application and elution of a biotinylated protein. Identical samples of biotinylated BSA (300 μg/1 ml Buffer A, pH 4) were applied to a 0.75 ml column of the Sepharose-nitro-avidin resin. The column was washed with Buffer A, pH 4, and eluted using Buffer C, pH 10. The procedure was repeated three additional times, and the fractions were monitored for protein by the Bradford assay (Fig. 1 1 A). As shown in Fig. 1 IB, accumulation of the eluted fractions gave essentially identical levels of biotinylated protein bound to and released from the nitro-avidin column per cycle. EXAMPLE 6. Purification of IgG on biotinyl-protein A/nitro-avidin column The performance of a nitro-avidin column as a universal affinity resin was examined In this approach, biotinylated protein A was attached to Sepharose and poured into a column, the column was used as an immunoaffinity column in the purification of immunoglobulin directly from whole rabbit serum, and the biotinylated protein A was subsequently released from the column To a 2-ml column containing 0 5 mg nitro-avidin/ml Sepharose prepared according to Example l(iv), 1 8 mg of protein A in 4 ml of Buffer A, pH 4, were added Whole rabbit serum (0 5 ml diluted 4-fold with 0 1 M Buffer B, pH 8) was applied to the column The column was washed with the same buffer, followed by the same buffer at 10 mM concentration The bound immunoglobulin was released from the column by Buffer A, pH 4 The biotinylated protein A was removed subsequently by 50 mM Buffer C, pH 10 The results are shown in Fig 12 The various peaks were then examined by SDS-PAGE, which indicated essentially pure fractions of the expected proteins (Fig 13) The purified immunoglobulin appeared to be as pure as a commercially available sample of an equivalent fraction, and the biotinyl protein A, which eluted from the column by alkaline treatment, was similarly pure EXAMPLE 7. Preparation of iodinated avidin and binding of biotinylated proteins to iodinated avidin adsorbed in microtiter plates (I) Preparation of iodinated avidin A sample of 2 mg avidin in 0.5 ml sodium phosphate buffer pH 7 was treated with 10 μl KI solution (32 mg/ml) and 200 μl of Chloramine-T (2 mg/ml) After incubation for 30 min at 23°C, 300 μl of sodium metabisulfite solution (2 mg/ml) were added for a period of 1 min Termination was performed by addition of 1 ml of 1% KI solution The sample was dialyzed overnight against double-distilled water (ii) Binding of biotinylated peroxidase to iodinated avidin The iodinated avidin was adsorbed to microtiter plates, the plates were blocked by a solution of 1% BSA, and samples of biotinylated horseradish peroxidase were applied in Buffers A, B and C of different pH (37 μg/0 1 ml buffer/well) The plates were washed and the peroxidase enzymatic activity was determined as described in method v(a) above using ABTS as a substrate As shown in Fig 14. optimal binding occurred at pH 5 EXAMPLE 8. Preparation of azotyrosine-avidin and streptavidin p-Arsanilic acid (p-aminobenzene arsonic acid) (100 mg ) dissolved in 0.3M HCl (10 ml) was treated in an ice-bath with NaNO 2 (35 mg in 5 ml water) After 6 min, the solution was adjusted to pH 5 with NaOH, and used immediately 0 5 ml of the resulting azoarsanilic acid (2 mg in 0 1 M sodium borate buffer, pH 8 4) was added to a solution of avidin (5 mg in 4 5 ml of 0 1 M sodium borate buffer, pH 8 4), and the reaction was carried out for 2 hours at room temperature The progress of the reaction was followed spectrophotometrically (λ naλ 342nm), and the azoarsanylate-derivatized avidin was dialyzed against PBS or 50 mM Tris buffer, pH 8 Azo-tyrosine streptavidin was prepared in a similar manner Substituting p-arsanilic acid by other p-aminobenzene derivatives, e g anthranilic acid (o-aminobenzoic acid), p-aminobenzoic acid, p-aminobenzenephosphoric acid and sulfanilic acid (p-aminobenzenesulfonic acid), the corresponding azo derivatives are obtained EXAMPLE 9. Preparation of aminotyrosine-avidin and streptavidin Nitro-avidin or azotyrosine-avidin (2 mg in 1 ml of 50mM Tris buffer, pH 8) was treated with a 24-fold molar excess of Na 2 S 2 O 4 (1 4 mg in 4 ml of the same buffer) The reaction was carried out at room temperature for 16 hours, and the extent of reduction was verified spectrophotometrically (decrease in absorbance at A-, ax 428nm for nitro-avidin or 342 nm for azotyrosine-avidin) The protein was dialyzed against PBS Aminotyrosine-streptavidin was produced by a similar procedure using 1 mg of nitro- streptavidin and a corresponding molar excess of Na 2 S 2 O REFERENCES 1 Argarana, C E , Kuntz, I D , Birken, S , Axel, R and Cantor, C R (1986) Nucl. Acids Res. 14, 1871 2 Bayer, E A , Ben-Hur, H and Wilchek, M (1990) Methods Enzymol. 184, 80 3 Bayer, E A and Wilchek, M (1990) Methods Enzymol. 184. 138 4 Bayer, E A and Wilchek, M (1992a) Methods in Molec.Biology JO, 137 5 Bayer, E A and Wilchek, M (1992b) Methods in Molec.Biology JO , 143 6 Chandra, G and Gray, G (1990) Methods Enzymol 184, 70 7 Finn, F M and Hofmann, K.H (1985) Methods Enzymol. 109. 418 8 Gitlin, G , Bayer, E A and Wilchek, M (1987) Biochem J. 242, 923 9 Gitlin, G Bayer, E A and Wilchek, M (1988a) Biochem J. 250, 291 10 Gitlin, G . Bayer, E A and Wilchek, M (1988b) Biochem. J 256, 279 11 Gitlin, G , Khaiy, I , Bayer, E A , Wilchek, M and Muszkat, K A (1989) Biochem. J 259, 493 12 Gitlin, G , Bayer, E A and Wilchek, M (1990) Biochem. J. 269, 527 13 Gorecki, M , Wilchek, M and Patchornik, A (1971) Biochim Biophys. Acta 229, 590-595 14 Green, N M (1975) Adv Protein Chem. 29, 85 15 Green, N M ( 1990 Methods Enzymol 184. 51 16 Hiller, Y . Bayer, E A and Wilchek, M (1990) Methods Enzymol. 184, 68 17 Katchalski-Katzir, E (1993) TIB TECH U, 471 18 Kohn, J and Wilchek, M (1984) Appl. Biochem Biotechnol 9, 285 19 Riordan. J F , Sokolovsky, M and Vallee, B L (1966) J. Am Chem. Soc. 88, 4104 20 Scott, J K (1992) TIBS H, 241 21 Sokolov sky, M , Riordan, J F and Vallee, B L (1967) Biochim Biophys Res. Commumc 27, 20-25 22 Suter, M Cazin, Jr J , Butler, J E and Mock, D M (1988) J Immunol. Meth 1 13, 83 23 Weber, P C , Ohlendorf, D H , Wendolosky, J J and Salemme, F R (1989) Science 243, 85 24.Wilchek, M. and Bayer, E.A. (1988) Analytical Biochemistry 171. 1. 25. Wilchek, M. and Bayer, E.A., Eds. (1990) "Avidin-Biotin Technology", Methods in Enzymology, Nol. 184, Academic Press, Inc., San Diego.

Claims: CLAIMS 1 A biotm-binding modified avidin-type molecule selected from the group of molecules comprising (I) native egg-white avidm, (n) recombmant avidm, (in) deglycosylated forms of avidm, (iv) bacterial streptavidin, (v) recombinant streptavidin, (vi) truncated streptavidin, and (vn) derivatives of (ι)-(vι) which are modified at sites other than the essential tyrosine residue, characterized in that in said biotin-binding modified avidm-type molecule the essential tvrosme residue in the biotm-bindmg site is modified in such a way that its pKa is decreased in compaπson to the pKa of the unmodified tyrosine residue in the corresponding unmodified avidin-type molecule 2 A. modified avidin-tvpe molecule according to claim 1, in which the pKa of the tv rosme in the biotin-binding site is decreased bv the addition of one or more electroonilic ancor nucleophihc groups on the t rosme residue 3 A. modified aviαin-type molecule according to claim 2, wherein tne modified essential tyrosine residue is of the formula wherein X, and X 2 are each a radical selected from nitro, halogen >TR,R 2 and -\=NR 3 m which Ri and R 2 are each selected from hvdrogen, C r C g alkvl and C r C 6 carooxyiic acyl, and R 3 is aryl substimted by an acidic radical Λ A modified avidm-type molecule accordmg to claim 3, in which X* anα/or X 2 is a nitro group 5 Nitro-tvrosine native egg-white avidm β Nitro-tyrosme bacterial streptavidin A method for the preparation of a biotin-bmding nitro-tyrosme modified avidm-type molecule according to anv one of claims 4 to 6, which comprises reacting the unmodified ldin- vpe molecule with tetranitromethane under nondenarurmg conditions S A modified avidm-type molecule accordmg to claim 3 in wnich X, aαd'or X 2 is -aiogen 9 A modified avidin-type molecule according to claim 8, m which said halogen is iodine 10 A modified avidm-type molecule according to claim 3, in which X ! and/or X 2 is an azo group 1 1 A modified avidin-type molecule according to claim 3 in wnich X, and/or X-, is an ammo group 12 A modified avidm-type molecule according to claim 3, .n which X* and/or X 2 is an — N=NR 3 group, in which R 3 is phenyl iubstituted by carboxvl or bv an acyl radical derived from an inorganic acid 13 A modified avidin-type molecule according to anv one of claims 1 to 6 and 8 to 12, attached to a solid suppoπ l ά A modified avidin-type molecule according to claim 13 wnerem the solid suppoπ is a resin, a microtiter plate, glass beads or magnetic beads 15 A modified avidm-type molecule according to claim 1- wnerem the solid suppoπ is a resin 16 A modified avidm-type molecule accordmg to claim 15, wherein the resm is Sepharose 17 A column for the immobilization of a biotinylated gand containing a modified avidin-type molecule of claim 1 attached to a resm 18 A column according to claim 17, wherein said modified avidin-type molecule is nitro-tyrosme native egg-white avidin and said resin is Sepharose 19 A column according to claim 17, wherem said modified avidin-type molecule is nitro -tyros ine bacterial streptavidin and said resm is Sepharose 20 Use of a biotm-bmding modified avidm-type molecule accordmg to any one of claims 1 to 6, 8 to 12 and 14 to 16, or of a column according to any one of claims 17 to 19, in a method employing the avidin-biotm technology 21 Use according to claim 20 of said modified avidm-type moiecule or said column in affimty chromatography, m cell separation, for cell immobilization and release, for the capture and release of DNA for the immobilization and release of biotinylated enzymes, for the production of phage libraries and as a reversiole matrix for biosensors 22 A process for the recov ery of either the avic in- column or * r.e oiotinvlated ligand in a method employing the av iαin-Diotm technology, whicn comprises (i) immobilizing a biotinylated ligand onto a column containing a modified avidm- type molecule according to claim 1 attached to a resin, (ii) carrying out a desired reaction or separation process with the thus immobilized biotinylated ligand, (iii) removing the biotinylated ligand from the immobilized modified avidm-column by altering the conditions, and (iv) recovering the biotinylated ligand and/or the modiπed avidin-column for further use 23 A process according to claim 22 wherein the biotinylated ligand is removed from the - n immobilized modified avidm-column by raising the pH, heating, adding excess concentrations of biotin or low concentrations of urea, guanidine or thiocyanate. and; or combinations thereof 24 A process according to claim 23 wherein the biotinyiated ligand is removed from the immobilized modified avidin-column by raising the pH to 10 I D 25 A process accordmg to claim 23 wherem the biotinylated ligand is removed from the immobilized modified avidin-column by adding 0 6 mM biotm 26 A process accordmg to any one of clauns 22 to 25 w nerem said modified avidin-type molecule has a modified essential tyrosine residue of the formula 25 wnerem X, and X 2 are each a radical selected from nitro, halogen, NR,R 2 and ~N=NR 3 m wnich R, and R 2 are each selected from hydrogen, C r C 8 alkyl and C r C d carboxylic acyl. and R 3 is aryl substimted by an acidic radical 27 A process according to any one of claims 22 to 25 wherem said modified avidin-type molecule is nitre -tyrosine native egg-white avidin 30 2S A process according to any one of ciaims 22 to 25 nerem said modified avidm-type moiecuie is nitre -tvrcsine bacterial streptavidin

Inventors: BAYER EDWARD A, MORAG ELY, WILCHEK MEIR, BAYER, EDWARD, A., MORAG, ELY, WILCHEK, MEIR
Applicants: BAYER EDWARD A, MORAG ELY, WILCHEK MEIR, YEDA RES & DEV, BAYER, EDWARD, A., MORAG, ELY, WILCHEK, MEIR, YEDA RESEARCH AND DEVELOPMENT CO. LTD.
Application year: 1996
Main classification: C12Q
IPC codes: B01J20, C07K14, C12N11, C12Q1, G01N30, G01N33