MXPA97010181A - Modified molecules of avidina and estreptoavidina and use of mis - Google Patents

Abstract

The present invention relates to modified biotin-binding avidin-type molecules wherein the essential tyrosine residue at the biotin-binding site is modified such that its pKa decreases in comparison to the pKa of the unmodified tyrosine residue at the corresponding unmodified avidin type molecule, the avidin-like molecules include: (i) native egg white avidin, (ii) recombinant avidin, (iii) deglycosylated forms of avidin, (iv) bacterial streptoavidin, (v) streptoavidin recombinant, (vi) truncated streptoavidin, and (vii) derivatives of (i) - (vi) that are modified in sites other than the essential tyrosine residue, the modification is achieved by substitution in one or both positions to the hydroxy radical of the Tyrosine residue by radicals such as nitro, halogen, azo and amino, modified avidin-like molecules can be used in all applications of avidin-bioti technology

Description

MODIFIED MOLECULES OF AVIDINR AND STREPTOAVIDINR AND USE OF THEMSELVES FIELD AND BACKGROUND OF THE INVENTION The present invention relates to a molecule of the avidin type that is modified at the tyrosome residue binding site, known to be critical for the binding of b? O +? N .. Modified avidin is still capable of binding hiotin or a ligand b? o + my side under specific conditions, but the alteration of these conditions, for example, high pH or competition with bio + in, removes the bound biotin or the ligand io my side . In this way, the invention provides a reversible form of avid for use in Avidin-bioin technology, thus "correcting" one of the main disadvantages of the aircraft molecule for various application purposes, namely, the conditions of denatural. extreme action required to interrupt the avidma-biotm complex. These drstic conditions necessary to associate the avidin-biotin complex usually and irreversibly inactivate the biological activity of the biotylated component, thus rendering it unsuitable for subsequent use. Avidin (tje the egg white) and estrep + oaví dina (from Streptornyce avidimi) are two related proteins that bind bio + in with similar dissociation constants of approximately 10-15 M igreen, 1975). In addition to the union to bio + m, many of its physical properties are quite similar. Both, for example, are four identical sub-domains fixed non-covalently, each of which has only a biotin site. The values of the subunit M are also very similar. In addition, several short stretches are conserved in the sequences of the two, par- ticularly stretchy proteins of Trp-Lys that occur in approximately similar positions (Aryarana et al., 1986). It was previously shown (Gitlin et al., 1907, 1988a) that certain residues of lis and tryptophan are involved in the biotin ion in both proteins (Gitlm et al., 1988b). More recently, it was shown that both avidin as this reptile exhibits the same three-dimensional folding, and that most of the junction site residues are identical or similar (Ueber et al., 1989). The geometry of the binding site and the junctions formed between both proteins with the biotm molecule are in fact very similar. Despite these similarities, there are several differences between the two proteins. Avidin is a bridged disulfide glycoprotein that contains two methionine residues, while streptoavi dyna is not glycosylated and is free of sulfur-containing amino acid side chains. Another significant difference is in the content of tyrosine. Avidin has only one tyrosine residue (Tyr-33), while streptoavidin has six tyrosine residues at positions 22, 43, 54, 60, 83 and 96. Interestingly, the only avidin tyrosine residue is located in a region containing a sequence with here Li of one of the tyrosine residues of this reptoavidin (Tyr-43 in the Thr-Gly-1 hr-iyr) .. This record of shots occupies a position prominent in the site of biot binding and the chemical modification of the hydroxyl group (Je ti osina leads to irreversible inactivation of the avidin molecule (Gitlin et al., 1990) Each monomer of avidi binds a molecule of biotm.

The unique feature of this union, then, is the effort and specificity of the formation of the avidin-biotm complex. The resulting affinity constant, calculated at 1.6 x I0i £ M-i for avidin and 2.5 x 1013 Mi for this reptoavidin (Green, 1990), is the highest known for a protein and an organic ligand. It is also so strong that biot m can not be released from the ion site, even when subjected to a variety of drastic conditions such as high concentrations of denatural agents at room temperature, for example, 6 M guanidimide hydrochloride, 3 M of guanidimo thiocyanate, 8 M of urea, 10% of β-rnercaptoethanol or 10% of sodium dodecyl sulfate. Ba or treatment cornbmado with guanidinio hydrochloride to pH ba or (1.5) or during the heating (>70 ° C) in the presence of denaturing agents or detergents, the protein is denatured; and biotin is evicted from the binding site i n < errurn i o. Avid recognizes biotin mainly in the ui-eido ring (urea type) of the molecule. The interaction between the avidin ion site and the sulfur-containing ring in the valeral chain of valeric acid in the mine is much less stressful. The relatively weak interaction between the chain later-al with carboxy biotin and avidin means that the former can be chemically modified and I-ijada a wide variety of biologically active material, -the portion of biotm of the derivative or conjugate resulting is still available for interaction with avid a. In turn, avidin can be derived with many other molecules, notably "probes" or different type registration groups. This is the mystery of avidin-biotm technology (Uilchek and Bayer-, 1990). In this way, 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 derived or conjugated with an appropriate probe. The use of avidin from egg white in avidi na-biot technology is sometimes restricted due to the high basic characteristic (pI-10.5) and the presence of sugar portions in the avidin molecule, which can lead to reactions not specific or otherwise unwanted. In recent years, bacterial protein, retinopathy, has largely replaced egg white avidin par-most applications in the avid a-hiot technology. However, the problems with streptoa i di na (a biotin-independent and high-cost cellular link) have prompted renewed interest in egg white avidin as the norm for avi dina-biot i n technology.

For this purpose, modified viruses that exhibit improved molecular characteristics both on the native pro-dina (and previous derivatives thereof) as well as on this virus have been prepared, such as avidins (eg, M-acyl). , for example, avidins of N-forrnyl, N-acetyl, and N-succirule These avian derivatives reduce the charge of the protein, but all are prepared by covalent attachment to the available avidin lyses, and the consequent blockage of free amino groups prevents the subsequent preparation of other types of conjugates (notably protein-protein conjugates such as avidin-labeled enzymes) which are often prepared by interlacing with lysine residues using bi-reactants. (For example, glutaraldehyde) A useful and effective alternative to lysine modification is modification by arginines.

In this case, the pl of the protein is efficiently reduced and the lysines are still available for subsequent interaction. Two different avidin derivatives that are modified in this way are commercially available. One, ExtrAv? DmR, can be obtained in various forms derived or conjugated functionally from igrna Chemical Cornpany (St. Louis, MO). One second, NeutraLite Av? D? NR (a product of Belovo 0 hern i ca 1, Ba s t o g ne, Bel g i c) se o d i f i ca < ? dem and which are to be purchased in lump sums. Although 1 < The reduction of the egg white ratio of one of the problems solves one of the problems, the presence of the oliyosacapdo residue remains as a serious source of non-specific interaction (independent of biotm) that resumes its application. The return of avidin from egg white to the norm pair-to avidi na -biotin technology has been contingent on the removal of its sugars. The possibilities of removing a sugar from a glycoprotein are quite limited; It is possible to do it chemistry or enzirnatj carnent e "The chemical methods available at present, for example, using HF or oxidation of p?? odato, are destructive or inefficient. The well-known enzymatic method, which employs N-glycanase (Tarentmo et al., 1984), is usually very expensive and not very effective for avidy when conventional methodology is used. Finally, a viable process for deglycosylation was established and the resulting product was subsequently modified from chemical inert by the argimnas and is known under the trademark NeutraLite Avidin * (Belovo Chemicals). Despite all these improvements, one of the main problems in the different applications of avi-dynabiot technology is the lack of reversibility of the union and the difficulty of separating the avidin and biotm portions at the end of the procedure. , without the denaturation of the avidma or damaging or making inactive the biological material that has been fixed by means of the biotm bridge. At the same time, it would be advantageous (particularly for industrial use) to remove damaged or inactive L material from the avid column, thus reconstituting the column for fixation of a new sample of the biot component of my side. An object of the present invention is to provide modified carriers that are still binding to biotm and can be used advantageously in methods employing avi-dma-biotin technology where the reversibility of the method is desired or is an advantage. The number of tyrosine residues in model peptides and proteins has been described using tetr-anit rornetano (Riordan et al., 1966; Sokolovsky et al., 1967). In a previous work of the present invention (Gitl et al., 1989), an avidin nitrotyros derivative was prepared by nitriding avidin from egg white dissolved in 9M urea with tet ramtrornetan (TNM). The resulting mtro-avidin preparation was rendered inactive, ie, it did not bind to biotm, since the nitration was carried out in a denatured form of avidin (in the presence of urea). The nitro-avidma thus prepared is completely unsuitable for use in the avidma-biot m technology. They have prepared avidma and est reptoavidma marked 125 l for analytical purposes. The only tyrosine residue from each location of avium is not accessible to iodination, Avidin becomes susceptible to iodination (method of (Lorarnin T) by the introduction of groups 3 - (p-hi droxifeml) propioni lo and This way, 125 T was prepared, marked with these groups, and it is also possible to use reagent Rol t on-Hunt.

I25? to mark avidma (Fmn and Hofrnann, 1985). Streptavidin 1251 was produced by the iodination of strept oavidin with Na125? using the iodogen method (Suter et al., 1988). Avidin and streptoav have not been described! It has unmarked iodines. The azotization and formation of shot residues a in peptides and model proteins have been previously described, for example, n-nuclease A (Gorecki et al., 1971; Sokolovsky et al., 196 A.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a modified avid to biotin type avid molecule selected from the group consisting of molecules comprising: (1) avidin from native egg white; (11) recumbentive avidma; (111) desglicoladas forms of avi i na; (iv) is bacterial reptoavidin; (v) recornbinant streptoavidin; (vi) truncated streptoav1din; and (vil) derivatives of (?) - (v?) that are modified at sites other than the essential tyrosome residue, characterized in that in said avidin-modified molecule from? nion to biotm the essential tyrosine residue at the binding site The biotm is modified in such a way that its pKa decreases in comparison with the pKa of the unmodified tyrosine residue in the corresponding unmodified avidin type molecule. The modified avidin or ti molecule of the invention has one or more electrophilic and / or nucleophilic groups in the essential tyrosome residue of the avidin-like molecule, and can be exemplified by the compounds wherein the modified tyrosine is of the formula : wherein Xi and 2 are each a radical selected from nitro, halogen, NRi R2 and -N = NR3 wherein Ri and R2 are selected from hydrogen, Ci-C and carboxylic acyl, and R3 is aryl substituted by an acid radical. In a preferred embodiment of the invention, the avidin-like molecule is modified by the addition of one or more electrophilic groups to the tyrosine residue. For example, avidin or streptoavidin can be modified by nitration or halogenation, preferably iodination, of the residue of atoms, as illustrated in Figure 1 (Xi or X2 is NO2 or halogen, preferably T), (This way reducing the pKa of the residue of i ros at the biotm binding site from 10.5-12.5 to 6.5-8.5, (Preference approximately 7.0) Said modi fi cation of the shot residue a in the avidin-like molecule involves the addition of one or electrophysical graphs in the ortho position (adjacent to the hydroxyl group) in the ring (Je shots) As an example of this type of modified avidma, the nitration of the tyrosine residue using tetranitromethane (TNM) was used. The avidma and streptoavidin that contains neither trotyrim resulting, hereinafter referred to as "nitro-aví dina" and "nit r'o-est reptoavidina", respectively, have been extensively studied in order to define relatively mild conditions for release the biotm portion or the biotin ligand side, for example, a biotmi antibody, enzyme, nucleic acid or cell. In the present invention, the analysis of avLdma was carried out under non-denaturing conditions, and it was shown that the resultant nitro-avionid and nitro-conata-to-a-day bind to a biotylated ligand in an effective and fair manner. at pH 4. When the pH rose to 8, the biotinylated ligand was still retained in the column, however, when the pH rose to 10, the biotyredylated ligand was released.Alternatively, at a lower pH (for example, between the pH scale of 4 to 8), the ligand bLoti i side can be released by exchange using free biot m. These characteristics of the ni ro-avidin and of the ni-ro-est reptoavidma provide forms of avidin and streptoavirus that are appropriate for a variety of applications.,. These materials have been used in accordance with the present invention to bind and subsequently release several examples of ligands from my sides to nitro-avidin and rut-strept oavidin immobilized in a ream of Sephar-os as well as for the fixation and release of biotinylated ligands to mt ro-avid and nitro-streptoavi dina adsorbed on rnicrotitle plates. In another embodiment, the present invention relates to avi dine or halogenated streptoavidin, rnuy preferred, unlabeled iodine. Iodination of the tyrosome residue in avidma or estr-eptoavidin with KI using the chloramine T procedure produced rnono- and / or diiodotyrosma-avidin or -streptoavidin, which also showed to be effective reversible forms of avidia. Another embodiment of the invention relates to molecules of the avidma type modified in the essential tyrosome residue by one or more core groups selected from NR1R2 and --N = NR3. In the azo derivatives of the invention, ie, the compounds wherein Xi and / or X is --N = NR3, R3 is aplo, preferably carbocyclic, most preferred, phenyl, substituted by an acid radical selected from carboxyl and a residue of an inorganic acid such as phosphoric, arsonic or suphonic acid. The azo derivatives according to the invention are prepared from the corresponding p-arnino derivatives, for example, p-arsanilic acid, anthranilic acid, p-aini nobenzoic acid, sterolfaric acid and p-alanic acid. also phosphorus-free, by diazotization with NaN2 and the reaction of the diazome salt with the molecule of the avidin-type of choice. In the amino acid derivatives (ie the invention, ie, the compounds in which Xi and / or X 2 is NRi 2, Ri and 2 ^ e are selected from H, alkyl and acyl, the alkyl radical is preferably? straight or branched Ci-Cß alkyl, examples being methyl, ethyl, propyl, isopropyl, butyl, hexyl, octyl The acyl radical is preferably a Ci-Cβ carboxylic acyl such as acetyl, yl, butyl, succinyl or benzyl icarbo ilo The arnmo derivatives of the invention can be prepared by reducing the corresponding azo derivatives with sodium hydrosulfite Na 2 SβO 4 or by reducing the corresponding nitro derivatives with N 2 fO «and, if desired, the arní group is not rented or acylated by normal methods. The modified avidma-like molecules of the present invention can be used in the different applications of the avid to biotin technology.

BRIEF DESCRIPTION OF THE DIBU30S Figure 1 shows a reaction scheme for the preparation of a modified α-amino-to-amino acid type of the invention and its use in a reversible method using the avi-biot i technology. Figure 2 shows levels (%) of avidin filtration as a function of the concentration (rnM) of t et ra it rnetantane (TNM), as described in example L (?). Figure 3 shows the effect of pH on the biotinylated bovine serum albumin (BSA) ion on a Sepharose-nitro-avion dina resin, as described in example l (?). Figure 4 shows the effect of pH on the binding of alkaline phosphatase bioinilated to plates (Je icrotite) which contains nitrous oxide-adsorbed nitro, as described in example 2 (ii), figure b shows the comparison between the biotin binding activity of avidin (closed circuits) and nitro-avidin (open circles), as described in Example 2 (???). Figure 6 shows the pH-induced release of biotimulated BSA from a ream of Sepharose-nitro-avidin, as described in Example 3 (?), Figure 7 shows the pH-induced release of biotilated BSA from a Sepharose-nitro-avidma resin, as described in Example 3 (FIG. l?).

Figure 8 shows the release of JSA biotylase from a ream of Sepharose-nit ro-avidin by competition with free biotin, as described in Example 3 (???). Figure 9 shows bioinduced induced elution of biotinylated BSA as a function of pH, as described in Example 3 (.v). The free b otin was dissolved in regulators of different pH, varying from 4 to 10. Elution at pH of 10 in the absence of biotin is shown for comparison. Figure 10 shows the release of biotin from blocked sites of nitro-avidin biotm, as described in example 4. Figures 11A-B show the results of the repeated application and elution of BSA biotmil from a nitro-avidin column. -avi ina- Sepharose, as described in Example 5. In Figure 11A, identical samples of biotreated BSA were applied successively (at a pH of 4 using Regulator A) to, and eluted (at a pH of 10 using Regulator C). ) from, a column containing a Sepharose-mtro-avidin resin. Figure 11B shows that the accumulation of the eluted fractions gave essentially identical levels of bound and eluted protein per cycle. Figure 12 shows the release of protein A biotini lac of a column containing a resin of Sepharose-nitro-avidin continuing with the purification of rabbit whole serum immunoglobulin, as described in example 6.

Figure 13 shows the SDS-PAGE profile of the coLinin samples in Figure 12; Line A, rabbit whole serum (applied fraction); Line B, column "flowing; Line E, commercial preparation of rabbit immunoglobulin; Line F, biotinylated protein A standard. Figure 14 shows the effect of pH on the release of biotilated horseradish peroxidase (Rmci-ot plates) which contain adsorbed iodinated avidma, as described in example 7.

DETAILED DESCRIPTION OF THE INVENTION The term "molecule (Jel type avidin" as used herein refers to the avidy of native egg white glycoprotein, to deglycosylated forms of avidin, to bacterial streptoavins produced by selected strains. < Je Strept oinyces, for example, St reptornyces avidinn, to truncated streptoavid- ma, and to avidin and streptoavi dina recornbmantes as well as to derivatives of native avi, degl icosi lada and recornbmante and native streptoaví dina, recornbi nante and truncated, which are modified in sites other than essential tyrosine, for example, N-acyl avid ace, for example, N-acetyl avidin, N-phthalyl and N-succimyl, and the commercial products ExtrAvidm ™ and Neutralite Avidin ™ "All forms (Mo molecules of the avidin type are encompassed by the present invention, both avidin and Ib They are native and rechargeable molecules, as well as derived molecules, for example, avids, not gil cosí ladas, aviclinas (Je N-adlo and est reptóvadi dinas t.) Some of these materials are available cornorcially, for example, avidma and estrept. oavi dma nativas, avidmas no gl i cosí lada < - av idinas de N-ací Lo y es reptoavi di na truncada, or can be prepared by well-known methods (see Green, 1990, for the preparation of avidin and streptococcus). i, Hiller et al., 1990, prepared the preparation of avid non-glycosylase Ada, Bayer- and others, 1990, for the preparation of this reptile and streptococcus avidin and streptoavirus. The recirculating materials can be prepared by normal recoinbi DNA techniques, for example, as described by Chandra and Gray, 1990, for avid recornbmante, and by Argarana et al., 1986, for streptoavid a reoornbi nant e. "Biotylated ligands" that can be used with modified avidins The methods of application (eg, the avidin biot technology) are bioti-forms or forms of desired ligands such as proteins, for example, antibodies, enzymes, lectins, or carbohydrates and conjugated gl i, e.g. glycoproteins, gangliosides, hepapna, polysaccharides, or nucleic acids, i.e., DNA and RNA, or phages, viruses, bacteria and other cells, wherein said ligands are covalently bound to biotm or to a homologous, analog or derivative of the same. Many biotimized ligands are available commercially or can be prepared by 1? normal methods (see, for example, Jiayer and Uilche, 1992a). Modified avidin-like molecules of the mvendon, mainly avidin derivatives and modified retinopathy at one or both positions ortho of the ion site (Je residue (Je tyrosine (see figure 1)), are suitable for reversible interaction with the portion of biotm, and in this way constitute important new tools for avi di-biot i technology. These modified avidin-like molecules allow to remove, under mild conditions, free biotm and / or biotylated ligands, for example, Biotylated enzymes and other biologically active biotylated materials, immobilized avid to ortho-phenol-modified molecules or soluble complexes comprising the avid modified with the biotinylated ligand in solution. excess of biotm, high pH, for example, pH of 10, relatively low non-denaturing bonds of urea, guanidine or thiocyanate, heat and / or combinations (ie the same.) In a preferred embodiment, the removal is carried out by adding an excess of biotin, - example, a solution of 0.6 rnM of biot passes through the modified avid column to displace the biotylated component The modified avidin-like molecules of the invention are suitable for use in any method employing the avidin-biotm technology , particularly in those methods where the reversibility of the amino-biotin ion is an advantage, for example, as columns of reversible immobilization for use in affinity chromatography for the removal of 11 affinity gands, to remove enzymes immobilized in this way creating a reversible enzyme r-ector; - to interrupt biological complexes soluble in solution consisting of a biotinylated material interlaced by an avidin bridge; pair to produce high affinity phage collections; and for use in cell separation, thereby facilitating the release of a viable or intact cell together with the recognition component or ligand, eg, antibody, from a resin, or to counteract the agglutination of said cells by disassociating the avidin bridge . The invention also relates to a molecule of the modified avidma type of the invention fixed to a solid support or matrix. Any solid support used in the art is suitable such as, but not limited to, ress, nickel plates, glass globules, magnetic beads and the like. The fixation of the avidm to the solid support can be covalent or non-covalent and is carried out by normal methods. In a preferred embodiment, the modified avidin-type molecule is immobilized in a resin, preferably Sepharose, and the affinity resin obtained from Sepharose-nitro-avirin can thus be poured into a column for isolation procedures (Bayer and Uilchek , 1992b). In the description herein the term "av d na-epharose column" will be used in a column containing a molecule of the modified avidma type of the invention immobilized on a resin Sepharose. These columns are useful par cularly for separation procedures. In another embodiment of the invention, the modified avidin-type molecule is fixed to grooves of nickel-plaque plates. In the well known affinity chromatography method, which is the prototype of all affinity methods, a binding ligand, e.g., an antibody or receptor, is attached to a solid support, such as Sepharose. This can be achieved by biotinylating said ligand, and by subsequent immobilization to Sepharose by an avidma or streptoavidin bridge. The resulting avi-di-Sepharose column is then used as a manipulation to isolate and purify material that interacts with said biot ligand on my Side. In many cases, it would be advantageous to separate the biotinylated ligand from the avidma-Sepharose column, either to recover the ligand itself, which may be precious or delicate in nature, or to reconstitute the column for alternative use. This can be achieved by using a column that contains a modified avidin-like molecule of the present invention to immobilize the biotinylated ligand, which can finally be released from the column by adding alkaline solutions (eg, Regulator C). , pH 10) or adding excess biot (for example, Ob inM any pH). A new biotinylated ligand or a fresh charge of the same biotinylated ligand can be added to the column of reconstituted DNA. The avi d? Na ~ b? O system? n has been used to separate cells by a variety of methods. One proposal is to use a biotinylated ligand (eg, antibody) that recognizes a cell surface molecule (eg, surface antigen). Can the biotinylated ligand be attached to a column? another type of matrix, such as magnetic beads, through an avidin bridge or idina treptoa. Alternatively, a suspension of a mixed cell population can be treated with the biotylated ligand and the cells having the surface molecule of interaction can be bound using avidin in solution. Commonly, this proposal has been used simply to selectively "remove" a given population of cells from the mixed population. " Once bound to the matrix or agglutinated by avid a, the interactions between the affinity involved (ie, between the biotinylated ligand and the surface molecule, and between avidin and biotm) can not be easily interrupted, in a way that would preserve cellular integrity or viability, for example, the interruption of the interaction between an antibody and an avidin usually requires, such as low pH, that are harmful to most cells. However, cells that do not interact can be recovered. In order to recover the bound cell population, a cell containing a modified avidma-like molecule of the present invention can be used and a solution than with Lene biotin (eg, 0.6 inM biotam or isotonic conditions, for example, 0.15 nm NaCl pH 7, or such excess concentrations of biotm in appropriate tissue culture medium) can be used to pair the cells (together with the biotylated ligand) of the column or cells Agglutinates can be dispersed using the same solution containing bio m. Enzymes immobilized in the food, pharmaceutical and chemical industries are used to a great extent (Katchalsky-Katzir, 1993). One of the problems with immobilized enzymes for use in enzyme reactor systems is that the enzyme or its matrix undergoes a <; I po of aging procedure. For example, enzymes are notoriously sensitive proteins, often have a definite half-life, in time, they can become inactive during use or storage. For industrial use, it would be advantageous for this machine to reuse the immobilized matrix once the bound enzyme is no longer useful. An enzyme reactor consisting of a biotinylated enzyme linked to a column containing an avid-modified molecule according to the present invention in this way can be reconstituted by removing the inactive biotinylated enzyme by the addition of alkaline solutions ( example, Regulator C, pH 10) or adding excess iotin (for example, 0.6 mM at any pH). A fresh charge of biotyped enzyme can then be added to the reconstituted modified avidin column. Similarly, the cells can be immobilized to a modified avidum column and used as a cell-based system. The cells can be released using a solution containing biotin (eg, 0.6 nm (Je biotm under isotonic conditions, for example, 0.15 M NaCl at a pH of 7, or such excess concentrations of biotin in a culture medium). suitable tissue), and the modified modified avidin column can be reconstituted in this manner.In the phage-collection technique, a? nion ligand (e.g., antibody, receptor) is attached to a solid support such as a plate This is often achieved by biotinylating said ligand, and by subsequent immobilization to the plate by means of a streptoavirus bridge, then bacteriophages with filament (for example, M13) can be added, and said phages, containing surface peptides that are capable of interacting with the immobilized ligand, thus binding to the plate.The non-specifically bound phages are removed by washing with low concentrations of neutral detergents such as tweet. in 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 in the phage. One of the potential problems in this pr-op is that some phages can still bind to plate 21 by means of affinity interactions very a. These phages would be of interest specifically due to their peptides of high affinity to the ligand biot my Side. From this invention, using the modified avidma or streptoavii of the present invention, high affinity phages can be released from the microtiter plates together with the bio-linked ligand, by the addition of alkaline solutions (e.g. Regulator-C, pH 10) or by adding excess biotin (for example, 0.6 rnM at any pH). the high affinity pep- tides removed attached to the phage can then be enriched by subsequent infection of bacteria, and by established phage collection procedures (see, eg, Scott, 1992). Gene enrichment and DNA isolation can be achieved by forming biotinylated DNA complex with a modified avidin type molecule or a modified avidin column according to the present invention by known methods (see Uilohek and Bayer, 1988). In the past, proteolytic enzymes, such as K protein, have been used to digest the avidin in order to release the biotinylated DNA from the complex. Using the modified avidma-type molecule of the present invention, the biotinylated DNA can be released using alkaline solutions (e.g., Regulator C, pH 10) or by adding excess biotm (e.g., 0.6 rnM at any pH). The biosensors consist of a detector-biological element that confers specificity and a translation function (that is, electrochemical, optical, calorimetric and acoustic) that converts a biological fact to a response that can be processed and quantified. The biological ligand may be catalytic (eg, enzymes, bacterial cells, tissues) or non-catalytic (eg, antibodies, receptors, DNA). These detectors depend on the immobilization of one of the components of interaction on the detection surface, and the resulting constituents (the biodetector apparatus ate "letad" are remarkably expensive.) The subject of avidin-biotm has been used in the past. The general method for immobilizing said ligands in biosensors The ability to replace the native avidin or streptoavidin with the avidins modified in accordance with the present invention would provide a reversible type of biosensor that would be an advantage in cost and convenience. In this manner, the biotinylated ligand can be immobilized to bLosensor by avidin or modified retinopathy, and when desired the biotinylated ligand can be released using solutions (eg, C-Regulator, pH 10). adding excess biotin (for example, 0.6 inM of biotm to any desired pH, ionic stress conditions, etc.) The modified avidma-like molecule of the biosensor it can be charged later with the biotylated ligand the same or different. The present invention also provides a method for the recovery of the column (Je avidin or the ligand biotmi Laclo in a method employing the avidin technoLogia-? bio m, (th) comprises: (i) mmobilizing a biotinylated ligand in α na ~ na containing a modified avidin-like molecule of the invention fixed to a resin; (11) carrying out a desired reaction or process. Separate the ligand with the hiotmylated ligand thus immobilized; (LIL) remove the ligand biotm Liado from the modified avidma column inrnoví 1 ized altering the conditions, and (ív) recover the ligand biotm liado and / or the modified avidin column for use The biotinylated ligand can be removed from the immobilized immobilized avid column by raising the pH, heating, adding excess concentrations of biotm, or low concentrations of urea, guamdine or thiocyanate, and / or combinations thereof. The biotinylated ligand is removed from the immobilized modified avidin column by raising the pH to LO, or by adding 0.6 mM biotin.The invention will now be illustrated by the following non-limiting examples.

EXAMPLES Materials and Methods (i) Materials. Avidma of egg white was provided by the STC laboratory (Uinnipeg, Canada) or Belovo Chemicals (Bastogne, Belgium). Streptoav Ldma was purified from culture filtrates of Streptornyces avidimi using a column of uni-noboth-Sopharose inejor-ada as previously described (Bayer- and others, 1990) ,, Sepharose 4B CL was from Pharmacia (Uppsala , Sweden). Tetranitromet anus was (Je Fluka, Protein A and bovine serum albumin (BSA) were from Sigina Chemical Co. (S. Louis, MO, USA). (11) pH Regulators: pH Regulator A: 50rnM citrate-phosphate, pH 3-6; Regula (Jorna pH B: HCl Tris 50 rnM, pH 7-9, and R-egulador- of pH C: sodium carbonate-HCl 50 mM, pH 10. (ni) Biotimulation procedures. The methods and enzymes used in the examples were biotyped by conventional biotylation methods using biotinyl N-hydroxysucciniride ester (BNHS) as previously described (Kohn et al.

Uilchek, 1990). dv) Immobilization of avidin and streptoavidin for Sepharose was carried out by the cyanogen bromide method as previously described (Kohn and Uilchek, 1984). (v) Enzyme assays. (a) Activity of but idasa de radbano. Peroxidase activity was determined using 2, 'azmo-b? D (3-ethylbenz-? Azol? -6-sulfonic acid) (ABTS) as substrate. The substrate solution included 2.5 rng of the substrate per 10 rnl in the pH regulator A, to which 10 ul of 30% hydrogen peroxide was added. Color formation was prescribed at 420 nrn. (b) The alkaline phosphate activity was determined using p-nitrofemlo phosphate as the substrate. The substrate application included 1 (1 g of the substrate dissolved in the pH regulator dietnolarm na IM (pH 9.8) q? Ec: ont? MgCl? 0.5 mM.The color formation- was given to 410 nm (vi) Protoma The protein was determined by the Bradford method using either avidma or retype (where appropriate) or HSA as a norm.

EXAMPLE 1 PREPARATION OF NITRO-AVIDINE, NITRQ-ESTREPTOAVIDINE AND ITS MOBILIZATION TO SEPHAROSE (i) Preparation of nor ro-aví di na. The samples of avidin declare of egg (5 rng in 1 rnl of regulator (Je pH tris 50 inM, at pH 8 or more) were treated with different concentrations of tetranit blunt anus (TNM) (0.5-5 μl cor- respon at about 5-50 nm), during 30 minutes at 23 ° C., samples were dialed at night, again against NaCl 1.

M and twice with water (sometimes distilled) As shown in figure 2, under the conditions of the modification procedure, the maximum levels (ca. 70%) of modification were reached using more than 20 nm concentrations of reagent, that is, an average of three of four subunits (Jel tetrarnero of avidin appeared to be modified.) In the following experiments, the nitro-avidin used was prepared using 2 μl. (11) Preparation of nitro-streptoavirane. The retinoavidin (2.5 mg per 1 i of pH regulator) was subjected to no traction using higher levels (Je tetra ni t romet no (6 μl corresponding to approximately 50 mM), due to a greater number of tyrosine residues. per subunit of the reptile organism (ni) Preparation (Je nit ro-aví dina and nitro-streptoavidin immobilized for Sepharose) (a) Activation of cyanogen bromide from Sepharose 4B CL. Se? Harose-4B CL drained first with water, then with 30% acetone (w / w) and finally with 60% acetone (w / w) The resin was resuspended in 6 rnl of 60% acetone and cooled to 0-4 ° C. While stirring with a magnetic stirrer, 6 rnl of CNBr solution (10 g / 100 i of acetone) was added followed by the dropwise addition of an identical volume of the triethylate to (TEA) (15.2 g / 100 ml of acetone) for a period of 1-2 minutes The activated ream is filtered and washed with 0.1 M sodium bicarbonate (b) nnoví pa ra tion Sepharose. the coupling of nitro-avidma and mtro-streptoavi dyna to Sephar-os was activated 4B CL re performed in a 0.1 M bicarbonate solution for 16 hours at 4 ° C. (iv) Nitration of avidma resins-Sepharose and streptoavidi na-Seph rose. A sample of 4 rnl of avidin-Sepharose ream (1.4 rng avidin / rnl Sepharose) prepared as described above (???) (b) but using unmodified avidin, was washed by means of Tris-regulator pH 50 rnM | > H 8, and treated with 6 μl of TNM for 50 minutes at 23 ° C. The resin modified with nitro was washed first with Na Cl 1 M, then with water twice distilled and finally with PBS.

EXAMPLE 2 LINK OF BIOTYNATED PROTEINS FOR NITRO-AVIDINE (i) The binding of biot thousand proteins for nitro-avLdine was tested in several ways. In one experiment, nitro-avidma was immobilized by Sepharose by the cyanogen bromide method (in accordance with the Km example) using approximately 0.5 ng of avidm per rnl of Sepharose, and samples of biotinylated BSA were applied to the regulator. pH A, B or C (100 μl) to 100 μl of the mtro-avirine Sepharose resin. The effluent fractions were measured for the protein. The percentage of binding in different pH values was determined by subtracting the amount of protein in the effluent fractions from those applied to the ream. As shown in Figure 3, the optimal bonding occurred at pH 4. The higher pH values (between 5 and 8), the levels of the bond plane were observed. Above pH 8, the bond dropped markedly and at pH 10, the bond was negligible. (n) Similar results were achieved using the biotinylated alkaline phosphatase enzyme in a rnicrotitration test plate (FIG. 4). In this experiment, the m-avidin prepared according to example (i) was adsorbed to rnicrotitration plates (1 μg or t-pyrimidine, 100 μl of phosphate-buffered saline solution (PBS / excavation) The plates were blocked by a solution (Je BSA at l%, and the samples of biotinylated alkaline phosphatase were applied in the pH regulators A, B or C of different pH (37 μg / 0.1 pH regulator / excavation). were washed or the binding enzyme fraction was determined by the enzyme assay as described in the above method (b) using p-nitrophenyl as a substrate. (111) Using a similar assay procedure of nitric avidin plate enzyme, the binding activity of nitro-avidin was compared with that of the native avidin (no rnodi ficad) "The rnicrotjtulation plates, coated with avidin or nitro-avidin (1 μg / 100 μl PBS / excavation), were loaded with difer concentrations Ingredients (between 10 ng and 1 μg in 150 μl of pH A buffer) of biotylated horseradish peroxidase at the optimum pH for experimentally determined binding (eg, pH 4). The plates were washed and the peroxidase enzyme activity was determined as described in the method v (a) above using ABTS as a substrate. As shown in Fig. 5, under these conditions, the realization of the biotin bond of the unmodified vic and the rut r-o-avidma can not be distinguished, EXAMPLE 3 RELEASE OF BIOTINILLATED PROTEINS FROM NITRO-AVIDINE (i) In order to determine the preferred conditions for the release (ie biotin of the mtro-avidma column), biotinylated BSA (1.5 mg / 150 μl regulator (Je pH A, pH 4) was applied to a column that contains a ream of nitro -avi dina-Sepharose according to example l dv.) The material bond was washed with pH regulators A, B or C of increasing pH, and the protein concentration of the effluent fractions was As shown in Figure 6, the alkaline solutions (regulator pH C, pH 10) were needed to release the biotinylated protein from the resin. (n) Using the same procedure, but only washing with regulator - of pH C, pH 10, similar results were obtained (Fig. 7) using a column of nitro-streptoavidin prepared by coupling mtro-streptoavidin to Sepharose according to the example Km). (m) In a similar experiment, the competition with free biotm was explored as a half-pair to release biotylated BSA from a column containing a rosin-avi-a-Sepharose resin prepared in accordance with Example Kiv), bLotinylated BSA (1.5 mg / 150 μl buffer pH A, pH 4) was applied to a ru-avi dma-Sepharose column of 2 ml. A solution containing 0.6 thousand bio in regulator of μH A in pH 4. l eluci n induced by biotLn of biot in-BSA was studied as a function of pH, using pH A, B or C regulators from pH 4 to pH 10. dv) A similar experiment was carried out under the same conditions with the res a nitro-avidi na-Sepharose prepared according to example l (? v), except that the free biotm was dissolved in pH regulator of different pH ranging from 4 to 10. As shown in Figure 9, the free biotm was an effective eluent over the entire tested pH scale. Elution at pH 10 in the absence of biotin will be compared.

EXAMPLE 4 BLOCKING LINK SITES OF NON-MODIFIED BIOTIN Because only partial modification of the binding site tyrosine could be achieved under the conditions described, unmodified sites could potentially be a problem in subsequent applications of nitro-avidin. In this way, the samples of nitro-avidin prepared according to the example Ki) containing different types of nitrous oxide (see Figure 2) were coated in the excavations of rni crotit ul plates. ation (1 μg / 100 μl PBS / excavation). The original avid egg white was used in similar amounts as control. The excavations were blocked with 1% BSA and the adsorbed protein samples were selectively blocked with excess levels of free bio m using 0.6 mM of bio + m in the pH regulator A. pH 4. The biotm that I occupy the modified binding sites could be released using alkaline solutions such as those described above in example 3 (pH regulator C, pH LO). After blocking and alkaline treatment, the binding capacity of IA biotm of the partially biotinylated avi dine samples was determined by the enzyme assay on rnicrotidy plates using the biotylated peroxidase system (method v (a) above). ). As shown in Fig. 10, the link was found to be proportional to the modification graph, with a maximum modification of approximately 60%.

EXAMPLE 5 REPEATED USE OF NITRQ-AVIDINE COLUMN The stability of the column containing a Sepharose-mtro-avidma ream according to the Km example) was proved by the repeated application and elution of a bio-laden protein. The identical samples of BSA biot muladas (300 μg / lrnl regulator-pH A, pH 4) were applied to a 0.75-milliliter column of the Sepharose-i t ro-avid a. The column was washed with regulator (pH A, pH 4, and eluted using the regulator (pH C, pH 10. the procedure was repeated three times, and the fractions were nominated to the protein by the Bradford test (figure HA) As shown in Figure IIB, the accumulation of eluted fractions gave essentially identical levels of the biotirulated protein bound to the mtro-avidin column and released therefrom per cycle.

EXAMPLE 6 PURIFICATION OF IgG IN BIOTINIL-PROTEIN A / NITRO-AVIDINE COLUMN The performance of a nitr-o-avidin column as a universal affinity resin was examined. In this approach, the biotinylated protein A was bound to Sepharose and poured into a column, the column was used as an immunoaffinity column in the purification of inrnunoglobulin directly from the rabbit whole serum, and the biotinylated protein was subsequently released from the spine. To a column of 2 ml containing 0.5 mg of rutro-avidin / rnl Sepharose prepared according to example ldv) 1.8 g of protein A in 4 ml of pH-regulator A, pH 4. was added. to the column the complete serum of rabbit (0.5 ml diluted times with 0.1 M regulator of pH 8, pH 8). The column was washed with the same pH regulator, followed by a pH regulator at a concentration of 10 in. The bound immunoglobulin was released from the column by means of the pH A regulator, pH 4. The A Biot my lame was subsequently removed by means of 50 nm of the pH C r-buffer, pH 10, The results are shown in Figure 12. The various peaks were then examined by SDS-PAGE, which indicated fractions. The purified immunoglobulins appeared to be as pure as a commercially available sample of an equivalent fraction, and the biotil A protein, which was extracted from the column by treatment. alkaline, it was even more pure.

EXAMPLE 7 PREPARATION OF YODINATED AVIDINE AND PROTEIN LINK BIOTINILLATES FOR YODINATED AVIDINE ADSORPED IN PLATES OF MICROTITULATION (I) Preparation of avid to yodi nothing. It was a sample of 2 rng from avi di na to 0. 5 rnl of regulated pH of sodium phosphate pH 7 with 10 μl of KI solution (32 mg / rnl) and 200 μl of <; lorarm na-T (2 ing / inl). After incubation for 30 minutes at 23 ° C, 300 μl was added to the solution of sodium abiotic solution (2 mg / ml) for a period of 1 in. The termination was repeated. The sample was dialyzed overnight with water distilled twice (n) Linkage of biotinylated peroxidase to avidin.

Adalid iodide was adsorbed to pair of labeling plates, the plates were blocked by a 1% BSA solution, and biotimized horseradish peroxidase samples were applied to pH regulators A, B, and C of different Ph (37 μl). /0.1 pH regulator / excavation). The plates were washed and the peroxidase enzyme activity was determined as described in the method v (a) above - using ABTS as a substrate. As shown in Figure 14, the optimal bond occurs at pH 5.

EXAMPLE 8 PREPARATION OF AZOTIROSINE-AVIDINE AND ESTREPTOAVIDINE 100 g of dissolved p-arsanilic acid was treated (p-arnmobencenarsomco acid) in 10 rnl of 0.3 M HCl in an ice barium with 35 ng of NaNÜ2 in 5 rnl of water. After 6 minutes, the solution was adjusted to pH 5 with NaOH and used immediately. 0.5 ml of the resulting azoarsamic acid (2 mg in pH-regulator 0.1 M sodium boronate, pH 8.4) was added to an avid solution (5 mg in 4.5 ml of pH 0.1 sodium borate buffer). M, pH 8.4) and the reaction was carried out during the hours at room temperature, the progress of the reaction was followed esperteppernent (\ max 342 nrn) and the avidin derived from azoarsam lato was dialyzed against PBS or regulator- ( The pH is 50 mM Tris, pH 8. The azo-t-ai-t-ai-t-ai-t-a-t-ai-i-t-ai-t-i-i-i-i-i-i-i-i-a-p-arsanilic acid has been prepared with other p-arnmobenzene derivatives, for example. anthranilic acid (o-ammobenzoic acid), p-arninobenzoic acid, acid pa inobenzene phosphorus and sulfanilic acid (p-arninobenzenesulfonyl acid), the derivatives are obtained (Je azo corresponding s.

EXAMPLE 9 PREPARATION OF AMINOTIROSINE-AVIDINE AND ESTREPTOAVIDINE They were treated (2 rng in 1 rnl regulator-pH Tris 50 rnM, pH 8) of nit ro-avidin or azotirosine with a molar excess of 24 times of Na2S2? «(1.4 rng in 4 rnl of the same pH regulator).

The reaction was carried out at room temperature for 16 hours and the degree of reduction was verified spectrophotometrically (decrease in adsorption at ν "x 428 nrn pair-a nitro-avidin or 342 nrn for azothyl-avidin). The protein was aliquoted against PBS. Arnmot i ros-na streptoav? Jna was produced by a similar procedure using 1 rng of nitro-strept oavid and a corresponding molar excess of Na? S20¿,.

REFERENCES I. Argarana, CE., K? Ntz, T.D., BirL-en, S., Axel, R. and Cantor, C.R. (1986) Nucí Acids Res j_4, 1871. 2. Bayer-, E.A., Ben-Hur-, H. and Uilehek, M. (1990) Methods Enzyrnol. 184, 80. 3. Bayer, E.A, and Uilchel- ', M. (1990) Methods Enzyrnol. 184, 138. 4. Bayer, E.A. and lilchel- ', M. (1992a) Methods in Molec. Biology 10, 137. 5. Bayer, E.A. and Uilchek, M. (1992b) llethods Molec. Biology 10, 143. 6. Chandra, G. and Gray, G. (1990) Methods Enzyrnol. 184, 70. 7. Finn, F.M. and Hofrnann, K.H. (1985) Methods Enzymol. 109, 418. 8. Gitlin, G., Bayer, E.A. and Uilchel- ', M. (1987) Biochern. 3. 242, 923. 9. Gitlm, G., Bayer, E.A. and Uilchek, M. (1988a) Biochem. J. 250, 291. 10. Gitlm, G., Bayer, E.A. and Uilchek, M. (1988b) Biochem. 3. 256, 279. II. Gitlm, G., Khaiy, I., Bayer, E.A., Ulilchek, M. and Muszkat, K.A. (1989) Biochern. 3. 259, 493. 12. Gitlin, G., Bayer, E.ñ. and Uilche, M. (1990) Biochem. 3. 269, 527. 13. Goreoki, M ", Uilchek, M. and Patchorni, A. (1971) Biochirn. Biophys. Acta 229, 590-595. L4. Gr-een, N.M. (1975) Adv. Protein Chern. _ '__., 85. 15. Green, N.M. (1990) Methods Enzymol. 18, 51. 16. Hiller, Y., Bayer, E.A. and Uilchek, M. (1990) Met hods Enzyrnol "184, 68. 17. Katchalski-Katzir, E. (1993) TIBTECH 11_, 471. 18. Kohn, 3. and Uilchek, M. (1984) Appl. Biochem. Biotechnol. _9, 285. 19. Riordan, J.F., Sokolovsky, M. and Val lee, B.L. (1966) 3. Am. Chern. Soc. 88, 4104. 20. Scott, J.K. (1992) TIBS 17, 241. 21. Sokolovsky, M., Riordan, 3.F. and Vallee, B.L. (1967) Biochirn. Biophys. Res. Cornrnunic. 27, 20-25. 22. Suter, M., Cazm, 3r. 3., Butler, J.E. and Mock, D.M. (1980) 3. Irnrnunol. Meth 113, 83. 23. Ueber, P.C., Ohlendorf, D.H., lendolosky, J.J. and Salernne, F.R. (1989) Science 243, 85. 24. Uilchek, M. and Bayer, E.A. (1988) Analytical Biochemistry 171, 1. 25. Uilchek, M. and Bayer-, E.A., Eds. (1990) "Avid -Biotm Technology", Methods in Fnzyrnology, vol. 184, Acadernic Press, Inc., San Diego.

Claims (1)

1. NOVELTY OF THE INVENTION CLAIMS 1. - A molecule of the avidin type modified from bioin selected from the group consisting of molecules that compose, (i) avidin from naive egg white; (n) recumbent avidin; (ni) desgli cosí 1 adas forms Je avi ina; (iv) is bacterial ept avii di; (v) streptoa i di na recornbi nante; (vi) est rept oavidi na truncada; and (vile) derivatives (Je (?) - (v?) Which are modified in sites differs from the essential tyrosome residue, characterized in that in said biotin-modified avidma-type molecule the essential tyrosine residue in the biotm-binding site it is modified in such a way that its pKa decreases in comparison with the pKa of the unmodified rosin residue in the corresponding unmodified avidin Lpo molecule 2. A modified avidma-type molecule according to claim 1, further characterized because the tyrosine pKa in the biotm binding site decreases by the addition of one or more electrophilic and / or nucleophilic groups in the tyrosine residue 3. A modified avidin-like molecule according to claim 2 , characterized further because the modified essential tyrosome residue is from the for-rnul: wherein Xi and X2 are each a radical selected from nitro, halogen, NR1R2 and --N = NR3 wherein Ri and R2 are selected from hydrogen, alkyl (Ci Ci and Ci carboxylic acyl) -Ce, and R3 is aplo subst? T ?? Jo by a radical aci (Jo.4- A modified avidin-type molecule according to claim 3, further characterized in that Xi and / or X is a nitro group. .- Native egg white avidma (Je nitro-ti rosina 6.- Bacterial streptoavidin nitro-thi rosma 7.- A method for the preparation of a molecule of the modified avidma type of mtro-t 11-os? Na of biotm binding according to any of claims 4 to 6, further characterized in that it comprises reacting the unmodified avidin-like molecule with tetranitronetan under non-denaturing conditions 8. A molecule of the modified avidin type according to claim 3, characterized further because Xi and / or X is halgene. 9.- One mol that of the modified avidma type according to claim 0, further characterized in that said halogen is iodine, 10. A molecule of the modified avidma type according to claim 3, further characterized in that Xi and / or X2 is an azo group. 11. A modified avidin-type molecule according to claim 3, further characterized in that Xi and / or X2 is an aromo group 12. A molecule of the modified avi-type according to claim 3, further characterized or &jue X Xi and / or X2 is a group --N = NR3, where R3 is phenyl substituted by oarboxyl or by an acyl radical derived from an inorganic acid 13. - A modified avidin type molecule ( In accordance with any of claims 1 to 6, and 8 to 12, further characterized in that it is fixed to a solid support 14.- A modified avidin-type molecule according to claim 13, further characterized because the solid support is a ream, a microtiter plate, glass globules or magnetic globules 15. A molecule of the modified avidma type according to claim 14, further characterized in that the solid support is a resin. - A molecule of the avidma type modified Co-orm ad with the claim 15, further characterized because the resin is Sepharose. 17. A column for the mobilization i a ligand bio i side containing a modified avidin type molecule in accordance with the rei fixed indication 1. A column according to claim 17, further characterized in that said modified avidma t-molecule is avian of native nitro-egg white and said resin is Sephaose.,. A column according to claim 17, further characterized in that said molecule (modified avidin-type Jel is a bacterial nitrate-N-osin bacterial ldi and said resin is Sepharose.) 20. Use of a modified aviclma type molecule. of binding to biotm according to any of claims 6, 8 to 12 and 14 to 16, or of a column of formulated with any of claims 17 to 19, in a method that implements the technology of avi d a -biot m 21. Use according to claim 20, further characterized in that said molecule (Jel modified avidma type or said column in affinity chromatography, in cell separation, for immobilization and cell release, for capture and release of DNA, for the immobilization and liberation of biotinded enzymes, for the production of phage collections and as a reversible matrix for biosensing 22.- A procedure for the recovery of the colu mna (Je avidin or ei ligand biot my side in a method employing the avi d? na -b? otm technology, which comprises: immobilizing- a biotinylated ligand in a column containing a modified avidma-like molecule of the invention fixed to a resin; (n) carry out a reaction (lesion or separation procedure with the biotylated ligand thus immobilized; (111) remove the bio-mined ligand from the immobilized modified avidin column by altering the conditions, and (iv) r-ecuperate the ligand A modified process according to claim 22, further characterized in that the biotinylated ligand is removed from the immobilized modified avidin column by raising the pH, heating, adding excess concentrations of biotm or low concentrations of urea, guamdma or thiocyanate, and / or combinations thereof 24. A method according to claim 23, further characterized in that the biotylated ligand is removed from the column of immobilized modified avidin raising the pH to 10. 25.- A method according to claim 23, further characterized in that the biotinylated ligand is removed from the c Modified immobilized av a modified olmnane adding 0.6 rnM of biotin. 26. - A method according to any of claims 22 to 25, further characterized by the fact that said modified avidin type molecule has a modified essential tyrosome residue of the formula: wherein Xi and X2 are each a radical selected from nitro, halogen, NRi R2 and -N = NR3 wherein Ri and R2 are selected from hydrogen, Ci-Ce alkyl and Ci-Cd carboxylic acyl , and R3 is aplo substituted by an acid radical. 27. A method according to any of claims 22 to 25, further characterized in that said modified avidin-like molecule is avidin of native egg clar-a nitro-tyrosine. 28. A method according to any of claims 22 to 25, further characterized in that said modified avidin-like molecule is bacterial streptoavidin of rutro-tyrosma.