WO1997000329A2 - Modified avidin and streptavidin molecules and use thereof - Google Patents
Modified avidin and streptavidin molecules and use thereof Download PDFInfo
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- WO1997000329A2 WO1997000329A2 PCT/IL1996/000014 IL9600014W WO9700329A2 WO 1997000329 A2 WO1997000329 A2 WO 1997000329A2 IL 9600014 W IL9600014 W IL 9600014W WO 9700329 A2 WO9700329 A2 WO 9700329A2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Definitions
- 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
- streptavidin from Streptomyces avidini ⁇
- bind biotin with similar dissociation constants of about 10 * M (Green, 1975).
- M Green, 1975
- the subunit M- values are also very similar.
- 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).
- Each avidin monomer binds one molecule of biotin.
- the unique feature of this binding 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.
- 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% ⁇ - mercaptoethanol or 10% sodium dodecyl sulfate.
- 6 M guanidinium hydrochloride 3 M guanidinium thiocyanate
- 8 M urea 10% ⁇ - mercaptoethanol or 10% sodium dodecyl sulfate.
- 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.
- 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).
- 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.
- 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.
- 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.
- 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 AvidinTM (a product of Belovo Chemicals, Bastogne, Belgium) is additionally modified and can be purchased in bulk quantities.
- Nitration of tyrosine residues in model peptides and proteins using tetranitromethane has been described (Riordan et al, 1966; Sokolovsky et al, 1967)
- a nitrotyrosine derivative of avidin was prepared by nitration of egg-white avidin dissolved in 9M-urea with tetranitromethane (TNM)
- TAM tetranitromethane
- 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.
- 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 ( ⁇ )-(v ⁇ ) which are modified at sites other than the essential tyrosine residue, cnaracte ⁇ zed 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 compa ⁇ son 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
- the avidm-type molecule is modified by addition of one or more electrophilic groups on the tyrosine residue
- 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 pos ⁇ t ⁇ on(s) (adjacent to the hvdroxvl grouD) on the tyrosine ring
- the nitration of the tvrosme residue using tetranitromethane (TNM) was employed as an example of this type of modified avidm.
- TAM tetranitromethane
- the nitration of avidin was performed under nondenatu ⁇ ng conditions, and the resultant nitro-avidin and nitro-streptavidin were shown to bind a biotinylated ligand efficiently and tightly at pH 4
- the pH was elevated to 8 the biotinylated ligand was still retained on the column
- 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
- 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
- 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-ammophospho ⁇ c acid, by diazotization with NaN0 and reaction of the resultmg diazomum salt with the avidm-type moecule of choice
- 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.
- 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.
- 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
- 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.
- 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
- 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.
- avidin-type molecule 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 ⁇ vidinii, 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 ExtrAvidinTM and Neutralite AvidinTM.
- N-acyl avidins e.g., N- acetyl, N-phthaiyl and N-
- 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
- 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
- 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
- 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
- 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 nondenatu ⁇ ng concentrations of urea, guanidine or thiocyanate, heat and/or combinations thereof
- 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
- 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, resin
- a binding hgand e g , an antibody or receptor
- a solid support such as Sepharose
- the resultant avidin-Sepharose column is then used as a handle to isolate and purify mate ⁇ al which interacts with said biotinylated ligand
- 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
- the avidin-biotin system has been used to separate cells by a va ⁇ etv of methods
- a biotinylated ligand e g , antibody
- the biotinylated ligand can be bound to a column or other type of matrix, such as magnetic beads, via an avidm or streptavidin bridge
- 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
- this approach has been used simply to selectively ' remove" a given population of cells from the mixed population Once bound to the mat ⁇ x 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
- I e affinity interactions involved
- 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 mat ⁇ x undergoes a type of agmg process
- 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
- 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
- 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 bioti ⁇ -containing solution (e g ,
- 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
- sucn excess concentrations of biotin in a suitable tissue-culture medium sucn excess concentrations of biotin in a suitable tissue-culture medium
- a binding ligand e.g., antibody, receptor
- 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
- 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.
- 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).
- alkaline solutions e.g., Buffer C, pH 10
- 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).
- proteolytic enzymes such as proteinase K
- 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).
- 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 )
- alkaline solutions e g , Buffer C, pH 10
- 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
- the present mvention also provides a process for the recovery of either the avidin- column cr the biotinylated ligand in a method employing the avi ⁇ in-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 avi ⁇ in-column by raising the pH, heating, adding excess concentrations of biotm, or low concentrations of urea, guanidine or thiocyanate and/or combmations
- EXAIVEPLE 1 Preparation of nitro-avidin. nitro-streptavidin and their immobilization to Sepharose
- 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 (l ⁇ g/lOO ⁇ lPBS/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 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 ⁇ l KI solution (32 mg/ml) and 200 ⁇ l of Chloramine-T (2 mg/ml) After incubation for 30 min at 23°C, 300 ⁇ l 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
- Azo-tyrosine streptavidin was prepared in a similar manner
- 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
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JP9502873A JPH11507920A (en) | 1995-06-14 | 1996-06-13 | Modified avidin and streptavidin molecules and uses thereof |
EP96918819A EP0871658B1 (en) | 1995-06-14 | 1996-06-13 | Modified avidin and streptavidin molecules and use thereof |
IL12244496A IL122444A (en) | 1995-06-14 | 1996-06-13 | Modified avidin and streptavidin molecules and use thereof |
DE69638257T DE69638257D1 (en) | 1995-06-14 | 1996-06-13 | MODIFIED AVIDINE AND STREPTAVIDINE AND METHODS OF USE THEREOF |
BR9609241-6A BR9609241A (en) | 1995-06-14 | 1996-06-13 | Modified avidin and streptavidin molecules and their use. |
US08/973,970 US5973124A (en) | 1995-06-14 | 1996-06-13 | Modified avidin and streptavidin molecules and use thereof |
AU61353/96A AU713417B2 (en) | 1995-06-14 | 1996-06-13 | Modified avidin and streptavidin molecules and use thereof |
AT96918819T ATE481417T1 (en) | 1995-06-14 | 1996-06-13 | MODIFIED AVIDIN AND STREPTAVIDIN AND METHODS OF USE THEREOF |
MXPA/A/1997/010181A MXPA97010181A (en) | 1995-06-14 | 1997-12-15 | Modified molecules of avidina and estreptoavidina and use of mis |
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IL11414995A IL114149A0 (en) | 1995-06-14 | 1995-06-14 | Modified avidin and streptavidin molecules and use thereof |
IL114149 | 1995-06-14 |
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WO1997000329A2 true WO1997000329A2 (en) | 1997-01-03 |
WO1997000329A3 WO1997000329A3 (en) | 1997-03-06 |
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US (1) | US5973124A (en) |
EP (1) | EP0871658B1 (en) |
JP (1) | JPH11507920A (en) |
KR (1) | KR19990022896A (en) |
CN (1) | CN1119353C (en) |
AT (1) | ATE481417T1 (en) |
AU (1) | AU713417B2 (en) |
BR (1) | BR9609241A (en) |
CA (1) | CA2221647A1 (en) |
CZ (1) | CZ401697A3 (en) |
DE (1) | DE69638257D1 (en) |
HU (1) | HUP9900239A3 (en) |
IL (1) | IL114149A0 (en) |
WO (1) | WO1997000329A2 (en) |
Cited By (11)
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US5985658A (en) * | 1997-11-14 | 1999-11-16 | Health Research Incorporated | Calmodulin-based cell separation technique |
EP0799890A3 (en) * | 1996-04-01 | 1999-12-22 | Roche Diagnostics GmbH | Recombinant inactive core streptavidin mutants |
EP0977770A1 (en) * | 1997-03-14 | 2000-02-09 | Trustees Of Boston University | Multiflavor streptavidin |
WO2000027814A1 (en) * | 1998-11-10 | 2000-05-18 | Yeda Research And Development Co. Ltd. | Avidin derivatives and uses thereof |
WO2000027813A1 (en) * | 1998-11-10 | 2000-05-18 | Yeda Research And Development Co. Ltd. | Azobenzene derivatives as labeling agents and intermediates thereof |
WO2000061790A1 (en) * | 1999-04-14 | 2000-10-19 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | High sensitivity phage display biomolecule detection |
WO2001009141A1 (en) * | 1999-07-29 | 2001-02-08 | National Institute Of Advanced Industrial Science And Technology | Polymerizable biotin derivatives, biotin polymer, and polymer responsive to avidin stimulation |
US6391571B1 (en) | 1997-04-01 | 2002-05-21 | Roche Diagnostics Gmbh | Recombinant inactive avidin mutants |
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US5863740A (en) * | 1994-03-05 | 1999-01-26 | Boehringer Mannheim Gmbh | Interference eliminating agent for application in immunoassays |
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1995
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-
1996
- 1996-06-13 AT AT96918819T patent/ATE481417T1/en not_active IP Right Cessation
- 1996-06-13 WO PCT/IL1996/000014 patent/WO1997000329A2/en not_active Application Discontinuation
- 1996-06-13 CA CA002221647A patent/CA2221647A1/en not_active Abandoned
- 1996-06-13 CZ CZ974016A patent/CZ401697A3/en unknown
- 1996-06-13 BR BR9609241-6A patent/BR9609241A/en not_active Application Discontinuation
- 1996-06-13 HU HU9900239A patent/HUP9900239A3/en unknown
- 1996-06-13 JP JP9502873A patent/JPH11507920A/en not_active Ceased
- 1996-06-13 KR KR1019970709365A patent/KR19990022896A/en not_active Application Discontinuation
- 1996-06-13 CN CN96195475A patent/CN1119353C/en not_active Expired - Fee Related
- 1996-06-13 US US08/973,970 patent/US5973124A/en not_active Expired - Lifetime
- 1996-06-13 AU AU61353/96A patent/AU713417B2/en not_active Ceased
- 1996-06-13 DE DE69638257T patent/DE69638257D1/en not_active Expired - Lifetime
- 1996-06-13 EP EP96918819A patent/EP0871658B1/en not_active Expired - Lifetime
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Also Published As
Publication number | Publication date |
---|---|
KR19990022896A (en) | 1999-03-25 |
AU713417B2 (en) | 1999-12-02 |
JPH11507920A (en) | 1999-07-13 |
HUP9900239A2 (en) | 1999-05-28 |
DE69638257D1 (en) | 2010-10-28 |
EP0871658A4 (en) | 2002-12-04 |
WO1997000329A3 (en) | 1997-03-06 |
US5973124A (en) | 1999-10-26 |
AU6135396A (en) | 1997-01-15 |
HUP9900239A3 (en) | 1999-11-29 |
MX9710181A (en) | 1998-03-31 |
CN1191018A (en) | 1998-08-19 |
IL114149A0 (en) | 1995-10-31 |
CA2221647A1 (en) | 1997-01-03 |
EP0871658B1 (en) | 2010-09-15 |
ATE481417T1 (en) | 2010-10-15 |
EP0871658A2 (en) | 1998-10-21 |
CZ401697A3 (en) | 1998-07-15 |
CN1119353C (en) | 2003-08-27 |
BR9609241A (en) | 1999-10-13 |
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