CA2198615A1 - Method of investigating the interaction of biomolecules using surface plasmon resonance - Google Patents
Method of investigating the interaction of biomolecules using surface plasmon resonanceInfo
- Publication number
- CA2198615A1 CA2198615A1 CA002198615A CA2198615A CA2198615A1 CA 2198615 A1 CA2198615 A1 CA 2198615A1 CA 002198615 A CA002198615 A CA 002198615A CA 2198615 A CA2198615 A CA 2198615A CA 2198615 A1 CA2198615 A1 CA 2198615A1
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- Prior art keywords
- peptide
- chelating agent
- group
- poly
- biosensor unit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
-
- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/26—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having more than one amino group bound to the carbon skeleton, e.g. lysine
Abstract
The invention concerns a process, a biosensor unit which can be regenerated and suitable kits for investigating the interaction between biomolecules by means of surface plasmon resonance (SPR). One of the reagents, a (poly)peptide, is coupled to the surface of the biosensor unit by means of a metal chelate. Nitrilotriacetic acid derivatives to which proteins with an affinity peptide containg histadine groups can be bonded are preferably used as chelate formers.
Description
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S015225J.65 Method of investigating the interaction of 5b;omolecules 1l~; ng surface plasmon resonance The invention relates to the investigation of the biospecific interaction of molecules.
In the past, analysis of the intermolecular 10interactions of macromolecules, for example (poly)peptide-(poly)peptide- or (poly)-peptide-DNA-interactions, generally too~ place under equilibrium conditions. Until recently, it was, with few exceptions, not possible, or only possible with difficulty, to investigate kinetic 15parameters because these investigations required the macromolecules to be present in relatively large quantities and at a relatively high degree of purity and/or because the available methods, such as af~inity chromatography or immunological methods, were not 20sufficiently rapid for monitoring biospecific interactions.
A method has recently been developed which is based on the optical phenomenon of surface plasmon resonance, SPR. The evaluation of optical signals which correlate 25with changes in the refractive index at the biospecific interface, and which are obtained in association with changes in the concentration of the macromolecules, which changes are elicited, for example, by the reaction partners binding to each other, now makes it possible to 30carry out this analysis in real time. In this method, smaller quantities of macromolecules, which do not carry any radioactive or fluorescent label, can be used and less stringent demands are placed on the purity of the macromolecule. This method has been disclosed, inter 35alia, in PCT applications WO 90/05295 and WO 90/05303.
The most widely employed, commercially available, system based on the surface plasmon resonance method -~ ~g8~13 (BIACoreTM, Pharmacia Biosensor) possesses a biosensor unit in the form of a so-called biosensor chip as one of its main elements in addition to the optical system and the sample transport equipment. The biosensor chip consists of a glass support which has a gold layer on one side to which layer a hydrogel matrix composed of carboxymethyl dextran is covalently bound by way of a barrier layer which is provided with linker groups. The hydrogel matrix is used, on the one hand, for immobilizing one of the reaction partners and, on the other hand, for providing the milieu which is required for analysing the biospecific interaction of the immobilized macromolecule with its reaction partner (Stenberg et al., 1991).
The following methods have so far been used to immobilize one of the reaction partners, which is generally a (poly)peptide, on the hydrogel matrix:
1) direct, irreversible immobilization on the hydrogel surface of the biosensor chip using chemical methods, 2) direct, irreversible immobilization of the biotinylated reaction partner on streptavidin or avidin which is bound to the hydrogel, and 3) indirect, reversible immobilization by means of binding the reaction partner by way of an antibody which is bound to the hydrogel.
However, these methods exhibit -a variety of disadvantages: Method 1) suffers from the risk that, due to the undirected chemical reaction between the hydrogel-forming substance and the (poly)peptide, which reactionpreferentially takes place, depending on the method employed, at primary amino groups, carbohydrate groups or free thiol groups of the (poly)peptide, the biological and/or biophysical properties of the latter are affected in a manner which either cannot be defined or can only be defined with difficulty. The consequence of this can be that the immobilized (poly)peptide is not, or is not ~9~ 3 com.pletely, in its native, biologically active form because, ~or example, the region of the molecule which is to interact with the reaction partner is blocked by a chemical group or has become inaccessible due to a confirmational change in the molecule.
Since the methods used for biotinylating macromolecules are basically similar to those for coupling (poly)peptides, these disadvantages, and conse~uently the methodological limitations, also apply to the method cited in 2). Both methods suffer ~rom the additional disadvantage that, although the ability to regenerate the hydrogel surface would constitute a substantial simplification of the method when carrying out serial analyses using the same (poly)peptide, it is very difficult to effect this regeneration while ret~;n;ng biological and biophysical activity.
These disadvantages can be avoided by using antibodies (method 3), when the same methods in principle are used as in sandwich ;mmlln~assays, for example in the ELISA (enzyme-linked ;mm1lnosorbent assay) technique. However, use of this method is limited by the need to have monoclonal, high-~ffln;ty antibodies available which are specific for the (poly)peptide. A further limitation is that the antibodies should not interact with epitopes which are of importance for the interaction with the reaction partners to be investigated.
The ;mmllnosensory converter for the surface plasmon resonance according to EP-A-0485874 comprises a carrier made of gold on the surface of which is a matrix of a m~nom~lecular intermediate layer and an immobilised ligand or antiligand layer.
This layer is two-~;m~n¢ional, unlike the three-~;m~n¢ional matrices based on carbohydrate.
The underlying object of the present invention was to provide a method for investigating the interaction of (poly)peptides with reaction partners by means of SPR.
Various methods are available for using recombinant DNA
methods to prepare proteins, whose sequences are elucidated, as fusion proteins having sequence segments which exhibit high affinity for a ligand (so-called ~ff;n;ty peptides). Immobilized metal chelate affinity chromatography (IMAC) is a method which is widely used for purifying proteins and peptides and in which such fusion proteins are bound to immobilized metal chelate~ by means of an a~finity peptide. Of the different chelating agents from the group of the iminodiacetic acid derivatives, nitrilotriacetic acid derivatives exhibit particularly advantageous properties including extremely high affinity for certain metal ions, for example Cu2+, Ni2+
or Zn2+. This method has hitherto been applied widely, using nickel as the metal ion and nitrilotriacetic acid as the complexing agent, for purifying recombinant fusion proteins to which an af~inity peptide having at least one histidine residue is bound directly or indirectly.
Methods of this nature for purifying recombinant proteins have been disclosed, inter alia, in European Patent Applications No. 184 355 (in which iminodiacetic acid is used as the complexing agent) and No. 282 042 (which describes nitrilotriacetic acid and proteins which posses~
an affinity peptide which has at least two adjacent histidine residues).
The concept of exploiting the principle o~ metal chelate a~inity chromatography in the SPR method formed the basis for achieving the object of the present invention.
The present invention consequently relates to a method for investigating the interaction of a (poly)peptide with a reaction partner using a biosensor unit in which the surface plasmon resonance which is elicited by the interaction is determined in a metallic layer at the interface of two media which are permeable to electromagnetic radiation and which are of differing refractive index, with the medium of lower refractive index being an aqueous medium in which the (poly)peptide is present in immobilized form and is brought into contact with the reaction partner.
The method is characterized in that the (poly)peptide is immobilized by way of a metal chelate.
In a further aspect, the invention relates to a $ ~ 3 biosensor unit for investigating the interaction of a (poly)peptide with a reaction partner by means o~
determining the ~ur~ace plasmon resonance in a metallic layer at the interface of two media which are permeable to electromagnetic radiation and which are o~ dif~ering refractive index, with the medium of lower refractive index being an aqueous medium. The biosensor unit is characterized in that a chelating agent, where appropriate in a form in which it i8 complexed with a metal ion, is bound to the surface of the biosensor unit which faces the aqueous medium.
In a preferred aspect, the aqueous phase is a biocompatible, porous matrix, in particular a hydrogel.
In principle, there is no limitation as regards the nature of the hydrogel formers, provided that they are basically suitable for use in the SPR method, particularly with regard to the requisite di~fusion of the biomolecules in the hydrogel matrix. Examples of suitable hydrogel formers are polysaccharides, such as agarose, dextran, carrageen, alginates, starch or cellulose, or derivatives of these polysaccharides, such as, for example, carboxymethyl derivatives, or water-swellable organic polymers, such as polyvinyl alcohol, polyacrylic acid, polyacrylamide or polyethylene glycol.
A particularly suitable hydrogel is one consisting of dextran, which, with regard to the covalent binding of the (poly)peptide, is provided with reactive groups, for example hydroxyl, carboxyl, amino, aldehyde, carbonyl, epoxy or vinyl groups. The design of the hydrogel layer, and its binding to the metal layer, which i9 effected, where appropriate, by way of an organic barrier layer, has been described, inter alia, in PCT application WO 90/05303 as well as by Lofas and Johnsson, 1990. In the preferred embodiment of the invention, the chelating agent is bound to the reactive groups of the hydrogel.
In a particularly preferred embodiment, the hydrogel former is a dextran which possesses carboxymethyl groups as reactive groups.
In an additional, preferred embodiment, the (poly)peptide is a fusion (poly)peptide which, in addition to its biologically active segment, possesses an affinity peptide which contains at least two adjacent histidine residues. In this case, the chelating agent is a nitrilotriacetic acid (NTA) derivative of the general formula Y-R-CH(COOH)-N(CH2COOH) 2 ~ wherein R may denote an alkylene bridge of the type (CH2) n~ which may be substituted or unsubstituted, with the proviso that the substituent does not have a detrimental effect on the function o~ the chelating agent, and n denotes an integer 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, whilst if the alkylene group is large enough it may possibly also contain one or more alkene or alkyne partial structures, or wherein R may denote an aromatic bridge member which may be made up of one or more mono- or polynuclear aromatic compounds which may optionally also be an aromatic heterocycle, or wherein R may denote an aralkyl bridge member in which the aromatic moiety may be bound to Y or to the ~-C atom adjacent to the carboxyl group, either directly or via an alkyl group of type (CH2) n~ ~ wherein n may denote an integer 1, 2, 3, 4 or 5, and wherein Y is a reactive group, particularly an NH2 or SH group.
Examples of R include methylene, ethylene, n-propylene or isopropylene and o-, m-, p-phenylene or naphthylene.
The NTA derivatives may also have the general formula Y-R1-CH(COOH)-N(CHR2COOH) 2 /
wherein R1 may denote a group having the meanings given for R, and wherein R2 may be an alkyl group of type CH3(CH2)n- which may be substituted, e.g. with OH or Cl, or unsubstituted, and n may denote an integer 1, 2, 3, 4 or 5, ~ 3 or wherein R2 may be a branched alkyl group such as isopropyl, t-butyl or isobutyl, or wherein R2 may be an aromatic bridge member having the meanings given for R, and wherein Y is a reactive group, particularly an NH2 or SH group.
R or Rl is selected such that, on the one hand, the complexing ability, as compared with that of the unbound NTA, is af~ected as little as possible and, on the other hand, such that the distance to the surface of the biosensor unit is sufficiently small not to interfere with the SPR phenomena.
R2 is selected such that the ability of the chelating agent to complex with the metal ion to be chelated and with the substance to be investigated is not negatively affected.
The suitability of substituents R or Rl or R2 can, for example, be tested by means of binding studies using a suitable ligand, as a rule a His(6)-modified polypeptide. Substituents which have a negative effect on the complexing ability reduce the affinity of the ligand; substituents which affect the nonspecific binding of the ligand increase the binding of the ligand for the chelator which is not complexed with a cation. Y is a reactive group by which the chelating agent is bound to the surface of the biosensor unit, in particular to the reactive groups which are contained in the hydrogel matrix. The reactive group of the chelating agent is conse~uently geared to the reactive groups of the surface of the biosensor unit, in particular of the hydrogel matrix; particularly preferably, the reactive group Y is an NH2 group which binds to the modi~ied carboxymethyl groups of the dextran. Other Y reactive groups which are suitable for the covalent bonding are SH groups which can be converted into a stable thioether bond. This thioether bond is superior to the disulphide bond with regard to stability in the presence of reducing agents, such as, for ~ ~ 9 ~
example, mercaptoethanol, which i8 frequently used in purifying or synthesizing (poly)peptides.
The chelating agent can be bound by way of its reactive yroups to the hydrogel using methods which are known for coupling (poly)peptides, for example using N-hydroxysuccinimide (NHS) and N-ethyl-N'-(dimethylamino-propyl)carbodiimide (EDC) (see, e.g., Cuatrecasas and Parikh, 1972).
Chelating agents which are suitable within the scope of the present invention have been described in US-A-4,877,830, EP-A-253 303 and WO 90/12803 and also in the papers of Hochuli and Piesecki, 1992, and Yokoyama et al., 1993,. Transition metal ions, preferably those of the fourth period are particularly preferred as the metal ions. Manganese, cobalt, nickel or copper ions are particularly preferred, especially Ni2+.
Within the scope of the present invention, N-(5-amino-l-carboxypentyl)iminodiacetic acid, which was described by Hochuli et al., 1987, and also N~,N~-di(l-carboxyethyl)-2,6-diaminohexanoic acid are particularly pre~erred a~ NTA derivatives, and nickel is particularly preferred as the metal ion which is complexed therewith.
With regard to the preparation of the fusion (poly)peptides, which exhibit at least two adjacent histidine residues for ensuring their ability to bind to the preferred metal chelate and which are used for the present invention in its preferred embodiment (so-called "His-Tag proteins"), reference is made to European Patent Application EP-A-282 042; examples of such His-Tag proteins are (His)6-proteins in which the affinity peptide possesses six histidine residues alongside each other.
In principle, it is also possible to couple the chelating agent directly to the metal surface of the biosensor chip, where appropriate by way of an organic barrier layer which possesses reactive groups. With regard to such barrier layers, reference is made to the disclosure in WO 90/05303. However, it is generally preferred to immobilize the (poly)peptide in the hydrogel matrix principally because the structure of the matrix corre~ponds quite well to the physiological conditions in the interior of the cell, thereby approximating the 5 natural environment for investigating the interaction of biomolecules.
The present invention possesses the crucial advantage that the biosensor surface is completely regenerable. This makes it possible to carry out serial experiments under comparable conditions. The novel biosensor unit fulfils the following demand~ which are placed on regeneration:
1) The (poly)peptide which is bound to the metal chelate can be completely removed.
2) On further loading, the surface of the biosensor unit does not lose any capacity for binding a (poly)peptide which i~ to be immobilized.
3) The binding properties of the immobilized (poly)peptide are unaffected.
Several options are available for regenerating a metal ion-saturated chelate surface which has bound a ligand, for example a His(6) protein. On the one hand, the ligand can be removed by acid treatment (e.g. 10 mM
acetic acid), the metal ion loading, depending on the metal ion employed, for the most part remaining stable.
If necessary, the bound protein, together with the metal ion, can be removed from the immobilized chelate bonding by adding another, strong chelating agent, such as, for example, 100 mM EDTA. The stability with which the metal ion is bonded to the chelating agent must be determined in each individual case and is dependent, inter alia, on the stability of the ligand/metal ion complex. When method reproducibility is taken into consideration, the method which uses other chelating agents to regenerate the chelate surface is to be preferred to the first method.
It is also necessary to test in each individual case whether regeneration of a sandwich-like surface, e.g.
~ t~
metal chelate-ligand 1-ligand 2 (where ligand 1 represents a (poly)peptide which has high affinity for the metal chelate surface, and ligand 2 represents a macromolecule which has affinity for ligand 1 but not for the metal chelate surface), can be achieved under conditions which permit removal of ligand 2 without affecting the binding of ligand 1 to the chelate surface. Solutions having high salt concentrations, as are used in classic affinity chromatography, can, in particular, be used for this purpose.
In addition to being used for the classical areas of application of the SPR method, the present invention can also be advantageously employed for biochemical purifications in order to verify fractions which contain the desired protein. This method is very greatly superior to the classical purification methods not only with regard to its precision and speed but also with regard to its simplicity.
In an additional aspect, the invention relates to a biosensor kit for investigating the interaction of a (poly)peptide with a reaction partner using SPR. The kit contains an SPR biosensor unit in a first container, a chelating agent in a further container, a salt of a metal which is suitable for complexing with the chelating agent in a further container, the reagents for activating the surface of the biosensor unit in one or more additional containers, where appropriate a reagent for deactivating the surface in another container, where appropriate a reagent for regenerating the surface in another container, and also, where appropriate, one or more comparison proteins in one or more additional containers.
Preferably, one surface of the biosensor unit consists of a hydrogel layer. Expediently, the chelating agent is in the form of a deep-frozen solution with the concentration and buffer solution being geared to the coupling to the surface of the biosensor unit.
Nitrilotriacetic acid derivatives, in particular N-(5-~g~5 amino-1-carboxypentyl)iminodiacetic acid and also N~,N~-di (1-carboxyethyl)-2,6-diaminohexanoic acid, are preferred chelating agents.
The metal salt nickel sulphate is preferred, preferably in the form of a stock ~olution which can be diluted depending on the desired degree of loading of the surface.
In the preferred embodiment of the invention, in which one surface of the biosensor unit consists of a hydrogel possessing reactive groups, in particular of a carboxymethylated dextran, the reagents for activating the biosensor surface are N-hydroxysuccinimi~e and N-ethyl-N'-(dimethylaminopropyl)carbodiimide which are preferably in the form of deep-frozen solutions which are suitable ~or the activation as regards concentration and buffer solution. In the preferred embodiment, the reagent for deactivating the N-hydroxysuccinimide groups which remain on the biosensor surface after coupling the (poly)peptide is ethanolamine, which is at suitable concentration and in a suitable buffer solution and is preferably likewise in deep-~rozen ~orm.
The reagent for regenerating the biosensor surface is pre~erably a chelating agent, in particular EDTA.
The test proteins which are optionally present in additional containers preferably possess an affinity peptide containing several histidine residues and are present as standard solutions at concentrations and in buffer solutions which are suitable for testing the loading of the biosensor surface.
In the experiments which were carried out within the scope of the present invention, the coupling was, in accordance with the preferred embodiment and also for the sake of simplicity, carried out in the hydrogel matrix because the commercially available biosensor units themselves have a dextran matrix and it appears that it is only with difficulty that this hydrogel layer can be - removed from the biosensor unit, as is required for the ~9~15 direct coupling, in a reproducible manner.
The coupling method using N-hydroxysuccinimide was employed to bind the chelating agents N-(5-amino-1-carboxypentyl)iminodiacetic acid or N~,N~-di(1-carboxyethyl)-2,6-diaminohexanoic acid to the biosensor hydrogel surface. In this method, the chelating agent, which contains a free primary amino group, was covalently coupled to the modified dextran hydrogel surface of a commercially available biosensor unit (BIACore). A
derivative of nitrilotriacetic acid which can be used directly for the immobilization was synthesized as the chelating agent. The derivative is synthesized in the main in accordance with the published method (Hochuli et al., 1987; and European Patent Specification 339 389), but with the difference that an acid cleavage using trifluoroacetic acid/trifluoromethanesulphonic acid was carried out, instead of the hydrogenation described, in the presence of palladium on charcoal, for eliminating the protective group. The material which is obtained in this way can, since it is to a large extent ~ree of inorganic impurities, be employed directly for coupling to the surface of the biosensor unit.
The chelating agent was immobilized using the method recommended by the biosensor chip manufacturer.
Since it is not possible to determine the immobilized quantity directly because of instrument limitations related to the low relative molecular weight of the nitrilotriacetic acid derivatives, the relative quantity was detected indirectly by means of determining the capacity for binding a protein. Commercially available bovine serum albumin (BSA) was found to be suitable for this purpose.
It was ascertained that the test (poly)peptide has very low affinity for the nickel-ion-free biosensor chip surface which has been modified with chelating agent; it was not possible to observe any binding of the (poly)peptide to the modified surface. The capacity of 98~5 ..
the surface to bind the test protein is, as shown in Example 4, correlated directly with the nickel ion concentration which is used for loading. By means of adjusting the nickel ion concentration, the binding capacity can be readily matched to the experimental re~uirements.
In an additional aspect, the present invention relates to the nitrilotriacetic acid derivative of the formula Na,N~-di(1-carboxyethyl)-2,6-diaminohexanoic acid.
This chelating agent can, like the known nitrilotriacetic acid derivatives, also be u~ed for immobilized metal chelate affinity chroma~ography for purifying proteins and peptides.
Table of Figures:
Fig. 1: Immobilization of N-(5-amino-1-carboxypentyl) iminodiacetic acid on the dextran surface of a biosensor unit Fig. 2: Indirect determination of the loading of the dextran sur~ace with N-(5-amino-1-carboxypentyl)-iminodiacetic acid using bovine serum albumin Fig. 3: Regeneration of the bovine serum albumin-loaded biosensor surfaces using EDTA
Fig. 4: Determination of the non-specific binding of a protein to the biosensor surface Fig. 5: Determination of the effect of the Nickel concentration on the ability to bind a protein to the biosensor surface Fig. 6: Ability to bind various proteins to the biosensor surface which is loaded with N-(5-amino-l-carboxypentyl) iminodiacetic acid/Ni2+
Fig. 7: Binding of a His(6)-modified protein to a biosensor surface which is loaded with Na,N~-di(1-carboxyethyl)-2,6-diaminohexanoic acid The invention is explained using the following examples:
~ ~19~
Example 1 a) Synthesis of N-(5-amino-1-carboxypentyl)iminodiacetic acid 4.17 g of bromoacetic acid (Aldrich) were dissolved in 15 ml o~ 2M NaOH and the ~olution was cooled to 0~C.
A solution of 4.2 g of N~-benzyloxycarbonyl-L-lysine (Fluka) in 22.5 ml of 2M NaOH wa~ slowly added to thi~
solution while stirring. After 2 hours, the solution was warmed to room temperature and then stirred overnight.
Subse~uently, the solution was heated at 50~c ~or 2 hour~
a~ter which 45 ml o~ lM HCl were slowly added to it. The resulting precipitate was centrifuged off, washed with 0.lM HCl and dried (5 g o~ product, theoretical yield 5.9 g). 0.87 g of the derivatives obtained above were dissolved in 10 ml of trifluoroacetic acid (Merck). 1 ml o~ tri~luoromethanesulphonic acid (Merck) was slowly added to the clear solution, which was being cooled on ice, and this mixture was then stirred at room temperature for 1 hour. The precipitate formed wa~ separated o~ and 30 ml of water were added to the solution, which was concentrated almost to dryness. An aliquot of this solution was fractionated on the PD10 column (Pharmacia) using water as the eluent. The ninhydrin-positive, neutral fractions were combined and lyophilized.
b) Synthesis o~ N~,N~-di(1-carboxyethyl)-2,6-diaminohexanoic acid 5.35 g o~ 2-bromopropionic acid (35 mmol) were dissolved in 15 ml of 2M NaOH and this solution was cooled to 0~C. A solution of 4.2 g of N~-benzyloxycarbonyl-L-lysine (15 mmol) in 22.5 ml o~ 2M NaOH was slowly added dropwise to this solution, at room temperature and while stirring, and the resultant mixture was ~tirred overnight at 70~C. 45 ml o~ lM HC1 were 810wly added to the solution after it had been cooled down. The precipitate was centrifuged off, washed with 0.lM HCl and dried. In order to eliminate the Z protective group, the precipitate was ~ ~19~6~
dis~olved in the minimum quantity of trifluoroacetic acid and a 0.1 volume of trifluoromethanesulphonic acid was added dropwise while cooling on ice. After having been stirred at room temperature for 1.5 h, the solution was added dropwise to 10 volumes of diethyl ether. The precipitate was washed 3x with ether and dried. The precipitate which had formed was separated off and 30 ml of water were added to the solution, which was concentrated almost to dryness. An aliquot of thi~
solution was fractionated on a PD10 column (Pharmacia) using water as the eluent. The ninhydrin-positive, neutral fractions were combined and lyophilized. The identity of the compound was confirmed by means of proton-NMR analysis.
Example 2 Coupling o~ N-(5-amino-1-carboxypentyl)iminodiacetic acid to the dextran surface of an SPR biosensor unit All the steps of the immobilization were carried out at 2soc in a sIAcOre device (Pharmacia) using Hss buffer (10 mM HEPES (2-[4-(2-hydroxyethyl)-1-piperazino]-ethanesulphonic acid), 150 mM NaCl and 5 mM MgCl2, pH
7.4)-A CM5 biosensor chip (Pharmacia biosensor, certified grade) was used for the immobilization. (This biosensor unit has a hydrogel surface which is composed of carboxymethylated dextran). The hydrogel surface was activated by injecting 35 ~1 of a 0.05 M N-hydroxy-succinimide (NHS)/0.2 M N-ethyl-N'-(dimethylaminopropyl)-carbodiimide (EDC) solution (flow rate 5 ~l/min). In order to couple the N-(5-amino-1-carboxypentyl)-iminodiacetic acid, which was described in Example 1, to the activated surface, 35 ~1 of a solution of 15 mg/ml N-(5-amino-1-carboxypentyl)iminodiacetic acid in lM NaOH
were injected at a flow rate of 3 ~l/min. Unsaturated binding sites on the surface were saturated by injecting ~ 2198~ ~
35 ~1 of a lM solution of ethanolamine (pH 8.5) (flow rate 5 ~l/min). In order to condition the surface, 10 ~l of a 100 mM solution of EDTA (adjusted to pH 8 with NaOH) were injected and the surface was loaded with 4 ~l of an 0. lM
NiSO4/o.2M CH3-COONa solution (flow rate 5 ~l/min). The course of this immobilization is depicted in Fig. 1 in term~ of the change in the surface plasmon resonance. The individual steps of the immobilization are indicated on the Figure. Since the loading of the surface with the chelating agent cannot be determined directly owing to the low relative molecular mass (Mr 295) of the agent, the loading was determined indirectly by injecting 35 ~l of a solution of 1 g of bovine serum albumin (BSA, Sigma) in 100 ml of running buffer at a constant flow rate (5 ~l/min). The sensorgram (Fig. 2) indicates significant binding of the protein to the surface. The reproducibility of the surface immobilization was confirmed by means of serial experiments using a standardized solution of this protein.
Example 3 Regenerability of the N-(5-amino-1-carboxypentyl) iminodiacetic acid surface In order to investigate the regenerability of the N-(5-amino-1-carboxypentyl)iminodiacetic acid surface, the following sequence of procedures was carried out 20 times in succession at a constant flow rate (5 ~l/min) and as described in Example 2: loading with Ni2+ ions, then injecting with 35 ~l of a solution of 1 g of bovine serum albumin (BSA, Sigma) in 100 ml of running buffer, and then regenerating with EDTA (ethylenediamine-N,N,N',N'-tetraacetic acid). In order to calculate the data - depicted in Fig. 3 (base line, injection m~; mllm and bound protein t=1), the resonance was determined 30 sec before injecting the bovine serum albumin (baseline), 30 sec before the end of the bovine serum albumin injection g~
(injection m~ m) and 30 sec after the bovine serum albumin in~ection (bound protein t=l).
EDTA (100 mM, pH 8) proved to be the most suitable reagent for removing the bound protein. As Fig. 3 shows, it is not possible to observe any loss in the activity of the surface on subsequent loading (injection maximum, bound protein t=l) or any retention of the bound protein following regeneration (baseline). Since EDTA removes the chelate-bound nickel ions, loading with Ni2+ ions must be carried out once again after the surface has been regenerated.
Example 4 Effect of the concentration of Ni2+ on the ability to bind protein In order to determine the nonspecific binding of the protein to the surface, the surface described in Example 1 was regenerated with EDTA, as described in Example 3, and 20 ~l of a solution of test protein was in]ected at a flow rate o~ 5 ~l/min. A~ter the sur~ace had been regenerated with EDTA once again, and loaded with 4 ~l of 0.1 M NiSo4/o.2M CH3-COONa solution (flow rate 5 ~l/min), a further 20 ~1 of the test protein solution were injected. As shown in Fig. 4, no binding to the surface can be observed in the absence of Ni2+;
however, after the sur~ace has been loaded with Ni2+, it can be seen to become saturated with test protein. In order to determine the effect of Ni2+ concentration on the binding ability of the surface, the Ni2+-N-(5-amino-l-carboxypentyl)iminodiacetic acid surface described in Example 1 was regenerated with EDTA, as described in Example 3, and loaded with in each case 4 ~l of an Ni2+
solution (O.lM NiS04/0.2M CH3-COONa, flow rate 5 ~l/min) of the given concentrations. After loading with the nickel had taken place, protein was injected and the resonance at t=l was determined. The plot of the resonance measured X1~61~
against the concentration of the nickel ion solution (Fig.
5) shows that complete loading of the surface is achieved using 4 ~l of a 100 ~M ~olution of Ni2+. Lower concentrations only result in incomplete loading of the surface, the extent of which depends on the concentration employed.
Example 5 The ability of different proteins to bind to the N-(5-amino-1-carboxypentyl) iminodiacetic acid surface In order to investigate the differing binding behaviour of different proteins, volumes of 40 ~l each of solutions (10 ~g/ml in running buffer) of a chick protein having the sequence depicted in the sequence listing, which sequence was modified with His(6) ((His)6-SCF), of bovine serum albumin (Sigma) and of egg white lysozyme (Sigma) were injected onto a biosensor surface which had been synthesized in accordance with Example 2. (The chick protein was prepared and purified following its expre~ion in a commercially available expression vector [pQE50, Diagen GmbH] in accordance with the purification protocol provided by the system manufacturer [The QIAexpressionist, Diagen GmbH, protocol 3, p. 35 and protocol 7, p. 45]. For its further purification, the protein was purified using an HQ/M anion exchange column [Perseptive Biosystems, Freiburg], and the protein concentration was determined by means of the Bradford protein assay [BioRad]). The binding behaviour of the individual proteins i8 depicted in Fig. 6: despite their relatively high concentrations, lysozyme (Mr 14300) and bovine serum albumin (Mr 68000) exhibit low resonance; by contrast, the His(6)-modified chick protein (Mr 22000) binds many times more strongly to the surface. It can also be seen that, in contrast to the His(6)-modified chick protein, lysozyme and bovine serum albumin achieve saturation of the surface. The surface which has been 19~15 loaded with the His (6) -modified chick protein to the point of saturation can be used to search for proteins, for example from the supernatants of chick cell line cell cultures, which interact with this protein by injecting cell culture supernatants from different cell lines. The binding of an interacting partner can be detected by the ampli~ication of the resonance which occurs following the injection.
Example 6 sinding of (His) 6-SCF to an N~ -di(1-carboxyethyl)-2,6-diaminohexanoic acid surface An N~,Na-di(1-carboxyethyl)-2,6-diaminohexanoic acid surface was prepared under exactly the same conditions as those described in Example 2 for N-(5-amino-1-carboxypentyl)iminodiacetic acid.
In order to investigate the behaviour of an (His) 6-modified protein when binding to this surface, the surface was loaded with Ni2+-ions, and 40 ~l of a solution (lo ~g/ml in running buf~er) o~ a chick protein having the His(6)-modified sequence ((His)6-SCF, described in Example 5) depicted in the sequence listing were injected onto this surface at a flow rate of 5 ~1/min. The sensorgram is depicted in Fig. 7. The surface shows a binding behaviour which is comparable to that of the surface which is modified with N-(5-amino-1-carboxypentyl)iminodiacetic acid and which is described in Example 2. The reproducibility of the surface immobilisation was confirmed by means of serial experiments using a ~tandardized solution of the protein.
- ~ ~19~
References Cuatrecasas, P. and Parikh, 1972, ~. Biochemi~try 11, 2291.
Hochuli, E., H. Dobel and Schacher, A., 1987, J.
Chromatography 411, 177-184.
Hochuli, E. and Piesecki, S., 1992, Methods 4 (San Diego), 66-72.
Lofas, S. and ~ohnsson, B., 1990, J. Chem. Soc., Chem.
Communications 1526.
Stenberg, E. et al., 1991, L. o~ Colloid and Interface Science, 143, 513.
Yokoyama, T., Sigeko, A. and Masatoshi, K., 1993, Chem.
Lett. 2, 383-386.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT
(A) NAME: Boehringer Ingelheim International GmbH
(B) STREET: Binger Strasse 173 (C) CITY: Ingelheim am Rhein (E) COUNTRY: FRG
(F) POSTAL CODE: 55216 (G) TELEPHONE: 06132/772282 (h) TELEFAX: 06132/774377 (ii) TITLE OF APPLICATION: Method for inve~tigating the interaction of biomolecules using surface plasmon resonance (iii) NUMBER OF SEQUENCES: 1 (iv) COMPUTER-READA;3LE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 204 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (vi) ORIGINAL SOURCE:
(A) ORGANISM: Gallus domesticus (G) CELL TYPE: fibroblast (ix) FEATURES:
(A) NAME/KEY: protein (B) LOCATION: 1..204 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
~ ~9~ 5 Met Ala Ser His His His Hi8 Hi8 His Gly Gly Ser Ala Gln Ser Ser Cys Gly Asn Pro Val Thr Asp Asp Val Asn Asp Ile Ala Lys Leu Val Gly Asn Leu Pro Asn Asp Tyr Leu Ile Thr Leu Lys Tyr Val Pro Lys Met Asp Ser Leu Pro Asn His Cys Trp Leu His Leu Met Val Pro Asp Phe Ser Arg Ser Leu His Asn Leu Leu Gln Lys Phe Ser Asp Ile Ser Asp Met Ser Asp Val Leu Ser Asn Tyr Ser Ile Ile Asn Asn Leu Thr Arg Ile Ile Asn Asp Leu Met Ala Cys Leu Ala Phe Asp Lys Asn Lys Asp Phe Ile Lys Glu Asn Gly His Leu Tyr Glu Glu Asp Arg Phe Ile Pro Glu Asn Phe Phe Ser Leu Phe Asn Ser Thr Ile Glu Val Tyr Lys Glu Phe Ala Asp Ser Leu Asp Lys Asn Asp Cys Ile Met Pro Ser Thr Val Glu Thr Pro Glu Asn Asp Ser Arg Val Ala Val Thr Lys Thr Ile Ser Phe Pro Pro Val Ala Ala Ser Ser Leu Arg Asn Asp Ser Ile Gly Ser Asn Thr Ser Ser Asn Ser Asn Lys Glu Ala Leu
-- TE~ PT ~ L ~ O
S015225J.65 Method of investigating the interaction of 5b;omolecules 1l~; ng surface plasmon resonance The invention relates to the investigation of the biospecific interaction of molecules.
In the past, analysis of the intermolecular 10interactions of macromolecules, for example (poly)peptide-(poly)peptide- or (poly)-peptide-DNA-interactions, generally too~ place under equilibrium conditions. Until recently, it was, with few exceptions, not possible, or only possible with difficulty, to investigate kinetic 15parameters because these investigations required the macromolecules to be present in relatively large quantities and at a relatively high degree of purity and/or because the available methods, such as af~inity chromatography or immunological methods, were not 20sufficiently rapid for monitoring biospecific interactions.
A method has recently been developed which is based on the optical phenomenon of surface plasmon resonance, SPR. The evaluation of optical signals which correlate 25with changes in the refractive index at the biospecific interface, and which are obtained in association with changes in the concentration of the macromolecules, which changes are elicited, for example, by the reaction partners binding to each other, now makes it possible to 30carry out this analysis in real time. In this method, smaller quantities of macromolecules, which do not carry any radioactive or fluorescent label, can be used and less stringent demands are placed on the purity of the macromolecule. This method has been disclosed, inter 35alia, in PCT applications WO 90/05295 and WO 90/05303.
The most widely employed, commercially available, system based on the surface plasmon resonance method -~ ~g8~13 (BIACoreTM, Pharmacia Biosensor) possesses a biosensor unit in the form of a so-called biosensor chip as one of its main elements in addition to the optical system and the sample transport equipment. The biosensor chip consists of a glass support which has a gold layer on one side to which layer a hydrogel matrix composed of carboxymethyl dextran is covalently bound by way of a barrier layer which is provided with linker groups. The hydrogel matrix is used, on the one hand, for immobilizing one of the reaction partners and, on the other hand, for providing the milieu which is required for analysing the biospecific interaction of the immobilized macromolecule with its reaction partner (Stenberg et al., 1991).
The following methods have so far been used to immobilize one of the reaction partners, which is generally a (poly)peptide, on the hydrogel matrix:
1) direct, irreversible immobilization on the hydrogel surface of the biosensor chip using chemical methods, 2) direct, irreversible immobilization of the biotinylated reaction partner on streptavidin or avidin which is bound to the hydrogel, and 3) indirect, reversible immobilization by means of binding the reaction partner by way of an antibody which is bound to the hydrogel.
However, these methods exhibit -a variety of disadvantages: Method 1) suffers from the risk that, due to the undirected chemical reaction between the hydrogel-forming substance and the (poly)peptide, which reactionpreferentially takes place, depending on the method employed, at primary amino groups, carbohydrate groups or free thiol groups of the (poly)peptide, the biological and/or biophysical properties of the latter are affected in a manner which either cannot be defined or can only be defined with difficulty. The consequence of this can be that the immobilized (poly)peptide is not, or is not ~9~ 3 com.pletely, in its native, biologically active form because, ~or example, the region of the molecule which is to interact with the reaction partner is blocked by a chemical group or has become inaccessible due to a confirmational change in the molecule.
Since the methods used for biotinylating macromolecules are basically similar to those for coupling (poly)peptides, these disadvantages, and conse~uently the methodological limitations, also apply to the method cited in 2). Both methods suffer ~rom the additional disadvantage that, although the ability to regenerate the hydrogel surface would constitute a substantial simplification of the method when carrying out serial analyses using the same (poly)peptide, it is very difficult to effect this regeneration while ret~;n;ng biological and biophysical activity.
These disadvantages can be avoided by using antibodies (method 3), when the same methods in principle are used as in sandwich ;mmlln~assays, for example in the ELISA (enzyme-linked ;mm1lnosorbent assay) technique. However, use of this method is limited by the need to have monoclonal, high-~ffln;ty antibodies available which are specific for the (poly)peptide. A further limitation is that the antibodies should not interact with epitopes which are of importance for the interaction with the reaction partners to be investigated.
The ;mmllnosensory converter for the surface plasmon resonance according to EP-A-0485874 comprises a carrier made of gold on the surface of which is a matrix of a m~nom~lecular intermediate layer and an immobilised ligand or antiligand layer.
This layer is two-~;m~n¢ional, unlike the three-~;m~n¢ional matrices based on carbohydrate.
The underlying object of the present invention was to provide a method for investigating the interaction of (poly)peptides with reaction partners by means of SPR.
Various methods are available for using recombinant DNA
methods to prepare proteins, whose sequences are elucidated, as fusion proteins having sequence segments which exhibit high affinity for a ligand (so-called ~ff;n;ty peptides). Immobilized metal chelate affinity chromatography (IMAC) is a method which is widely used for purifying proteins and peptides and in which such fusion proteins are bound to immobilized metal chelate~ by means of an a~finity peptide. Of the different chelating agents from the group of the iminodiacetic acid derivatives, nitrilotriacetic acid derivatives exhibit particularly advantageous properties including extremely high affinity for certain metal ions, for example Cu2+, Ni2+
or Zn2+. This method has hitherto been applied widely, using nickel as the metal ion and nitrilotriacetic acid as the complexing agent, for purifying recombinant fusion proteins to which an af~inity peptide having at least one histidine residue is bound directly or indirectly.
Methods of this nature for purifying recombinant proteins have been disclosed, inter alia, in European Patent Applications No. 184 355 (in which iminodiacetic acid is used as the complexing agent) and No. 282 042 (which describes nitrilotriacetic acid and proteins which posses~
an affinity peptide which has at least two adjacent histidine residues).
The concept of exploiting the principle o~ metal chelate a~inity chromatography in the SPR method formed the basis for achieving the object of the present invention.
The present invention consequently relates to a method for investigating the interaction of a (poly)peptide with a reaction partner using a biosensor unit in which the surface plasmon resonance which is elicited by the interaction is determined in a metallic layer at the interface of two media which are permeable to electromagnetic radiation and which are of differing refractive index, with the medium of lower refractive index being an aqueous medium in which the (poly)peptide is present in immobilized form and is brought into contact with the reaction partner.
The method is characterized in that the (poly)peptide is immobilized by way of a metal chelate.
In a further aspect, the invention relates to a $ ~ 3 biosensor unit for investigating the interaction of a (poly)peptide with a reaction partner by means o~
determining the ~ur~ace plasmon resonance in a metallic layer at the interface of two media which are permeable to electromagnetic radiation and which are o~ dif~ering refractive index, with the medium of lower refractive index being an aqueous medium. The biosensor unit is characterized in that a chelating agent, where appropriate in a form in which it i8 complexed with a metal ion, is bound to the surface of the biosensor unit which faces the aqueous medium.
In a preferred aspect, the aqueous phase is a biocompatible, porous matrix, in particular a hydrogel.
In principle, there is no limitation as regards the nature of the hydrogel formers, provided that they are basically suitable for use in the SPR method, particularly with regard to the requisite di~fusion of the biomolecules in the hydrogel matrix. Examples of suitable hydrogel formers are polysaccharides, such as agarose, dextran, carrageen, alginates, starch or cellulose, or derivatives of these polysaccharides, such as, for example, carboxymethyl derivatives, or water-swellable organic polymers, such as polyvinyl alcohol, polyacrylic acid, polyacrylamide or polyethylene glycol.
A particularly suitable hydrogel is one consisting of dextran, which, with regard to the covalent binding of the (poly)peptide, is provided with reactive groups, for example hydroxyl, carboxyl, amino, aldehyde, carbonyl, epoxy or vinyl groups. The design of the hydrogel layer, and its binding to the metal layer, which i9 effected, where appropriate, by way of an organic barrier layer, has been described, inter alia, in PCT application WO 90/05303 as well as by Lofas and Johnsson, 1990. In the preferred embodiment of the invention, the chelating agent is bound to the reactive groups of the hydrogel.
In a particularly preferred embodiment, the hydrogel former is a dextran which possesses carboxymethyl groups as reactive groups.
In an additional, preferred embodiment, the (poly)peptide is a fusion (poly)peptide which, in addition to its biologically active segment, possesses an affinity peptide which contains at least two adjacent histidine residues. In this case, the chelating agent is a nitrilotriacetic acid (NTA) derivative of the general formula Y-R-CH(COOH)-N(CH2COOH) 2 ~ wherein R may denote an alkylene bridge of the type (CH2) n~ which may be substituted or unsubstituted, with the proviso that the substituent does not have a detrimental effect on the function o~ the chelating agent, and n denotes an integer 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, whilst if the alkylene group is large enough it may possibly also contain one or more alkene or alkyne partial structures, or wherein R may denote an aromatic bridge member which may be made up of one or more mono- or polynuclear aromatic compounds which may optionally also be an aromatic heterocycle, or wherein R may denote an aralkyl bridge member in which the aromatic moiety may be bound to Y or to the ~-C atom adjacent to the carboxyl group, either directly or via an alkyl group of type (CH2) n~ ~ wherein n may denote an integer 1, 2, 3, 4 or 5, and wherein Y is a reactive group, particularly an NH2 or SH group.
Examples of R include methylene, ethylene, n-propylene or isopropylene and o-, m-, p-phenylene or naphthylene.
The NTA derivatives may also have the general formula Y-R1-CH(COOH)-N(CHR2COOH) 2 /
wherein R1 may denote a group having the meanings given for R, and wherein R2 may be an alkyl group of type CH3(CH2)n- which may be substituted, e.g. with OH or Cl, or unsubstituted, and n may denote an integer 1, 2, 3, 4 or 5, ~ 3 or wherein R2 may be a branched alkyl group such as isopropyl, t-butyl or isobutyl, or wherein R2 may be an aromatic bridge member having the meanings given for R, and wherein Y is a reactive group, particularly an NH2 or SH group.
R or Rl is selected such that, on the one hand, the complexing ability, as compared with that of the unbound NTA, is af~ected as little as possible and, on the other hand, such that the distance to the surface of the biosensor unit is sufficiently small not to interfere with the SPR phenomena.
R2 is selected such that the ability of the chelating agent to complex with the metal ion to be chelated and with the substance to be investigated is not negatively affected.
The suitability of substituents R or Rl or R2 can, for example, be tested by means of binding studies using a suitable ligand, as a rule a His(6)-modified polypeptide. Substituents which have a negative effect on the complexing ability reduce the affinity of the ligand; substituents which affect the nonspecific binding of the ligand increase the binding of the ligand for the chelator which is not complexed with a cation. Y is a reactive group by which the chelating agent is bound to the surface of the biosensor unit, in particular to the reactive groups which are contained in the hydrogel matrix. The reactive group of the chelating agent is conse~uently geared to the reactive groups of the surface of the biosensor unit, in particular of the hydrogel matrix; particularly preferably, the reactive group Y is an NH2 group which binds to the modi~ied carboxymethyl groups of the dextran. Other Y reactive groups which are suitable for the covalent bonding are SH groups which can be converted into a stable thioether bond. This thioether bond is superior to the disulphide bond with regard to stability in the presence of reducing agents, such as, for ~ ~ 9 ~
example, mercaptoethanol, which i8 frequently used in purifying or synthesizing (poly)peptides.
The chelating agent can be bound by way of its reactive yroups to the hydrogel using methods which are known for coupling (poly)peptides, for example using N-hydroxysuccinimide (NHS) and N-ethyl-N'-(dimethylamino-propyl)carbodiimide (EDC) (see, e.g., Cuatrecasas and Parikh, 1972).
Chelating agents which are suitable within the scope of the present invention have been described in US-A-4,877,830, EP-A-253 303 and WO 90/12803 and also in the papers of Hochuli and Piesecki, 1992, and Yokoyama et al., 1993,. Transition metal ions, preferably those of the fourth period are particularly preferred as the metal ions. Manganese, cobalt, nickel or copper ions are particularly preferred, especially Ni2+.
Within the scope of the present invention, N-(5-amino-l-carboxypentyl)iminodiacetic acid, which was described by Hochuli et al., 1987, and also N~,N~-di(l-carboxyethyl)-2,6-diaminohexanoic acid are particularly pre~erred a~ NTA derivatives, and nickel is particularly preferred as the metal ion which is complexed therewith.
With regard to the preparation of the fusion (poly)peptides, which exhibit at least two adjacent histidine residues for ensuring their ability to bind to the preferred metal chelate and which are used for the present invention in its preferred embodiment (so-called "His-Tag proteins"), reference is made to European Patent Application EP-A-282 042; examples of such His-Tag proteins are (His)6-proteins in which the affinity peptide possesses six histidine residues alongside each other.
In principle, it is also possible to couple the chelating agent directly to the metal surface of the biosensor chip, where appropriate by way of an organic barrier layer which possesses reactive groups. With regard to such barrier layers, reference is made to the disclosure in WO 90/05303. However, it is generally preferred to immobilize the (poly)peptide in the hydrogel matrix principally because the structure of the matrix corre~ponds quite well to the physiological conditions in the interior of the cell, thereby approximating the 5 natural environment for investigating the interaction of biomolecules.
The present invention possesses the crucial advantage that the biosensor surface is completely regenerable. This makes it possible to carry out serial experiments under comparable conditions. The novel biosensor unit fulfils the following demand~ which are placed on regeneration:
1) The (poly)peptide which is bound to the metal chelate can be completely removed.
2) On further loading, the surface of the biosensor unit does not lose any capacity for binding a (poly)peptide which i~ to be immobilized.
3) The binding properties of the immobilized (poly)peptide are unaffected.
Several options are available for regenerating a metal ion-saturated chelate surface which has bound a ligand, for example a His(6) protein. On the one hand, the ligand can be removed by acid treatment (e.g. 10 mM
acetic acid), the metal ion loading, depending on the metal ion employed, for the most part remaining stable.
If necessary, the bound protein, together with the metal ion, can be removed from the immobilized chelate bonding by adding another, strong chelating agent, such as, for example, 100 mM EDTA. The stability with which the metal ion is bonded to the chelating agent must be determined in each individual case and is dependent, inter alia, on the stability of the ligand/metal ion complex. When method reproducibility is taken into consideration, the method which uses other chelating agents to regenerate the chelate surface is to be preferred to the first method.
It is also necessary to test in each individual case whether regeneration of a sandwich-like surface, e.g.
~ t~
metal chelate-ligand 1-ligand 2 (where ligand 1 represents a (poly)peptide which has high affinity for the metal chelate surface, and ligand 2 represents a macromolecule which has affinity for ligand 1 but not for the metal chelate surface), can be achieved under conditions which permit removal of ligand 2 without affecting the binding of ligand 1 to the chelate surface. Solutions having high salt concentrations, as are used in classic affinity chromatography, can, in particular, be used for this purpose.
In addition to being used for the classical areas of application of the SPR method, the present invention can also be advantageously employed for biochemical purifications in order to verify fractions which contain the desired protein. This method is very greatly superior to the classical purification methods not only with regard to its precision and speed but also with regard to its simplicity.
In an additional aspect, the invention relates to a biosensor kit for investigating the interaction of a (poly)peptide with a reaction partner using SPR. The kit contains an SPR biosensor unit in a first container, a chelating agent in a further container, a salt of a metal which is suitable for complexing with the chelating agent in a further container, the reagents for activating the surface of the biosensor unit in one or more additional containers, where appropriate a reagent for deactivating the surface in another container, where appropriate a reagent for regenerating the surface in another container, and also, where appropriate, one or more comparison proteins in one or more additional containers.
Preferably, one surface of the biosensor unit consists of a hydrogel layer. Expediently, the chelating agent is in the form of a deep-frozen solution with the concentration and buffer solution being geared to the coupling to the surface of the biosensor unit.
Nitrilotriacetic acid derivatives, in particular N-(5-~g~5 amino-1-carboxypentyl)iminodiacetic acid and also N~,N~-di (1-carboxyethyl)-2,6-diaminohexanoic acid, are preferred chelating agents.
The metal salt nickel sulphate is preferred, preferably in the form of a stock ~olution which can be diluted depending on the desired degree of loading of the surface.
In the preferred embodiment of the invention, in which one surface of the biosensor unit consists of a hydrogel possessing reactive groups, in particular of a carboxymethylated dextran, the reagents for activating the biosensor surface are N-hydroxysuccinimi~e and N-ethyl-N'-(dimethylaminopropyl)carbodiimide which are preferably in the form of deep-frozen solutions which are suitable ~or the activation as regards concentration and buffer solution. In the preferred embodiment, the reagent for deactivating the N-hydroxysuccinimide groups which remain on the biosensor surface after coupling the (poly)peptide is ethanolamine, which is at suitable concentration and in a suitable buffer solution and is preferably likewise in deep-~rozen ~orm.
The reagent for regenerating the biosensor surface is pre~erably a chelating agent, in particular EDTA.
The test proteins which are optionally present in additional containers preferably possess an affinity peptide containing several histidine residues and are present as standard solutions at concentrations and in buffer solutions which are suitable for testing the loading of the biosensor surface.
In the experiments which were carried out within the scope of the present invention, the coupling was, in accordance with the preferred embodiment and also for the sake of simplicity, carried out in the hydrogel matrix because the commercially available biosensor units themselves have a dextran matrix and it appears that it is only with difficulty that this hydrogel layer can be - removed from the biosensor unit, as is required for the ~9~15 direct coupling, in a reproducible manner.
The coupling method using N-hydroxysuccinimide was employed to bind the chelating agents N-(5-amino-1-carboxypentyl)iminodiacetic acid or N~,N~-di(1-carboxyethyl)-2,6-diaminohexanoic acid to the biosensor hydrogel surface. In this method, the chelating agent, which contains a free primary amino group, was covalently coupled to the modified dextran hydrogel surface of a commercially available biosensor unit (BIACore). A
derivative of nitrilotriacetic acid which can be used directly for the immobilization was synthesized as the chelating agent. The derivative is synthesized in the main in accordance with the published method (Hochuli et al., 1987; and European Patent Specification 339 389), but with the difference that an acid cleavage using trifluoroacetic acid/trifluoromethanesulphonic acid was carried out, instead of the hydrogenation described, in the presence of palladium on charcoal, for eliminating the protective group. The material which is obtained in this way can, since it is to a large extent ~ree of inorganic impurities, be employed directly for coupling to the surface of the biosensor unit.
The chelating agent was immobilized using the method recommended by the biosensor chip manufacturer.
Since it is not possible to determine the immobilized quantity directly because of instrument limitations related to the low relative molecular weight of the nitrilotriacetic acid derivatives, the relative quantity was detected indirectly by means of determining the capacity for binding a protein. Commercially available bovine serum albumin (BSA) was found to be suitable for this purpose.
It was ascertained that the test (poly)peptide has very low affinity for the nickel-ion-free biosensor chip surface which has been modified with chelating agent; it was not possible to observe any binding of the (poly)peptide to the modified surface. The capacity of 98~5 ..
the surface to bind the test protein is, as shown in Example 4, correlated directly with the nickel ion concentration which is used for loading. By means of adjusting the nickel ion concentration, the binding capacity can be readily matched to the experimental re~uirements.
In an additional aspect, the present invention relates to the nitrilotriacetic acid derivative of the formula Na,N~-di(1-carboxyethyl)-2,6-diaminohexanoic acid.
This chelating agent can, like the known nitrilotriacetic acid derivatives, also be u~ed for immobilized metal chelate affinity chroma~ography for purifying proteins and peptides.
Table of Figures:
Fig. 1: Immobilization of N-(5-amino-1-carboxypentyl) iminodiacetic acid on the dextran surface of a biosensor unit Fig. 2: Indirect determination of the loading of the dextran sur~ace with N-(5-amino-1-carboxypentyl)-iminodiacetic acid using bovine serum albumin Fig. 3: Regeneration of the bovine serum albumin-loaded biosensor surfaces using EDTA
Fig. 4: Determination of the non-specific binding of a protein to the biosensor surface Fig. 5: Determination of the effect of the Nickel concentration on the ability to bind a protein to the biosensor surface Fig. 6: Ability to bind various proteins to the biosensor surface which is loaded with N-(5-amino-l-carboxypentyl) iminodiacetic acid/Ni2+
Fig. 7: Binding of a His(6)-modified protein to a biosensor surface which is loaded with Na,N~-di(1-carboxyethyl)-2,6-diaminohexanoic acid The invention is explained using the following examples:
~ ~19~
Example 1 a) Synthesis of N-(5-amino-1-carboxypentyl)iminodiacetic acid 4.17 g of bromoacetic acid (Aldrich) were dissolved in 15 ml o~ 2M NaOH and the ~olution was cooled to 0~C.
A solution of 4.2 g of N~-benzyloxycarbonyl-L-lysine (Fluka) in 22.5 ml of 2M NaOH wa~ slowly added to thi~
solution while stirring. After 2 hours, the solution was warmed to room temperature and then stirred overnight.
Subse~uently, the solution was heated at 50~c ~or 2 hour~
a~ter which 45 ml o~ lM HCl were slowly added to it. The resulting precipitate was centrifuged off, washed with 0.lM HCl and dried (5 g o~ product, theoretical yield 5.9 g). 0.87 g of the derivatives obtained above were dissolved in 10 ml of trifluoroacetic acid (Merck). 1 ml o~ tri~luoromethanesulphonic acid (Merck) was slowly added to the clear solution, which was being cooled on ice, and this mixture was then stirred at room temperature for 1 hour. The precipitate formed wa~ separated o~ and 30 ml of water were added to the solution, which was concentrated almost to dryness. An aliquot of this solution was fractionated on the PD10 column (Pharmacia) using water as the eluent. The ninhydrin-positive, neutral fractions were combined and lyophilized.
b) Synthesis o~ N~,N~-di(1-carboxyethyl)-2,6-diaminohexanoic acid 5.35 g o~ 2-bromopropionic acid (35 mmol) were dissolved in 15 ml of 2M NaOH and this solution was cooled to 0~C. A solution of 4.2 g of N~-benzyloxycarbonyl-L-lysine (15 mmol) in 22.5 ml o~ 2M NaOH was slowly added dropwise to this solution, at room temperature and while stirring, and the resultant mixture was ~tirred overnight at 70~C. 45 ml o~ lM HC1 were 810wly added to the solution after it had been cooled down. The precipitate was centrifuged off, washed with 0.lM HCl and dried. In order to eliminate the Z protective group, the precipitate was ~ ~19~6~
dis~olved in the minimum quantity of trifluoroacetic acid and a 0.1 volume of trifluoromethanesulphonic acid was added dropwise while cooling on ice. After having been stirred at room temperature for 1.5 h, the solution was added dropwise to 10 volumes of diethyl ether. The precipitate was washed 3x with ether and dried. The precipitate which had formed was separated off and 30 ml of water were added to the solution, which was concentrated almost to dryness. An aliquot of thi~
solution was fractionated on a PD10 column (Pharmacia) using water as the eluent. The ninhydrin-positive, neutral fractions were combined and lyophilized. The identity of the compound was confirmed by means of proton-NMR analysis.
Example 2 Coupling o~ N-(5-amino-1-carboxypentyl)iminodiacetic acid to the dextran surface of an SPR biosensor unit All the steps of the immobilization were carried out at 2soc in a sIAcOre device (Pharmacia) using Hss buffer (10 mM HEPES (2-[4-(2-hydroxyethyl)-1-piperazino]-ethanesulphonic acid), 150 mM NaCl and 5 mM MgCl2, pH
7.4)-A CM5 biosensor chip (Pharmacia biosensor, certified grade) was used for the immobilization. (This biosensor unit has a hydrogel surface which is composed of carboxymethylated dextran). The hydrogel surface was activated by injecting 35 ~1 of a 0.05 M N-hydroxy-succinimide (NHS)/0.2 M N-ethyl-N'-(dimethylaminopropyl)-carbodiimide (EDC) solution (flow rate 5 ~l/min). In order to couple the N-(5-amino-1-carboxypentyl)-iminodiacetic acid, which was described in Example 1, to the activated surface, 35 ~1 of a solution of 15 mg/ml N-(5-amino-1-carboxypentyl)iminodiacetic acid in lM NaOH
were injected at a flow rate of 3 ~l/min. Unsaturated binding sites on the surface were saturated by injecting ~ 2198~ ~
35 ~1 of a lM solution of ethanolamine (pH 8.5) (flow rate 5 ~l/min). In order to condition the surface, 10 ~l of a 100 mM solution of EDTA (adjusted to pH 8 with NaOH) were injected and the surface was loaded with 4 ~l of an 0. lM
NiSO4/o.2M CH3-COONa solution (flow rate 5 ~l/min). The course of this immobilization is depicted in Fig. 1 in term~ of the change in the surface plasmon resonance. The individual steps of the immobilization are indicated on the Figure. Since the loading of the surface with the chelating agent cannot be determined directly owing to the low relative molecular mass (Mr 295) of the agent, the loading was determined indirectly by injecting 35 ~l of a solution of 1 g of bovine serum albumin (BSA, Sigma) in 100 ml of running buffer at a constant flow rate (5 ~l/min). The sensorgram (Fig. 2) indicates significant binding of the protein to the surface. The reproducibility of the surface immobilization was confirmed by means of serial experiments using a standardized solution of this protein.
Example 3 Regenerability of the N-(5-amino-1-carboxypentyl) iminodiacetic acid surface In order to investigate the regenerability of the N-(5-amino-1-carboxypentyl)iminodiacetic acid surface, the following sequence of procedures was carried out 20 times in succession at a constant flow rate (5 ~l/min) and as described in Example 2: loading with Ni2+ ions, then injecting with 35 ~l of a solution of 1 g of bovine serum albumin (BSA, Sigma) in 100 ml of running buffer, and then regenerating with EDTA (ethylenediamine-N,N,N',N'-tetraacetic acid). In order to calculate the data - depicted in Fig. 3 (base line, injection m~; mllm and bound protein t=1), the resonance was determined 30 sec before injecting the bovine serum albumin (baseline), 30 sec before the end of the bovine serum albumin injection g~
(injection m~ m) and 30 sec after the bovine serum albumin in~ection (bound protein t=l).
EDTA (100 mM, pH 8) proved to be the most suitable reagent for removing the bound protein. As Fig. 3 shows, it is not possible to observe any loss in the activity of the surface on subsequent loading (injection maximum, bound protein t=l) or any retention of the bound protein following regeneration (baseline). Since EDTA removes the chelate-bound nickel ions, loading with Ni2+ ions must be carried out once again after the surface has been regenerated.
Example 4 Effect of the concentration of Ni2+ on the ability to bind protein In order to determine the nonspecific binding of the protein to the surface, the surface described in Example 1 was regenerated with EDTA, as described in Example 3, and 20 ~l of a solution of test protein was in]ected at a flow rate o~ 5 ~l/min. A~ter the sur~ace had been regenerated with EDTA once again, and loaded with 4 ~l of 0.1 M NiSo4/o.2M CH3-COONa solution (flow rate 5 ~l/min), a further 20 ~1 of the test protein solution were injected. As shown in Fig. 4, no binding to the surface can be observed in the absence of Ni2+;
however, after the sur~ace has been loaded with Ni2+, it can be seen to become saturated with test protein. In order to determine the effect of Ni2+ concentration on the binding ability of the surface, the Ni2+-N-(5-amino-l-carboxypentyl)iminodiacetic acid surface described in Example 1 was regenerated with EDTA, as described in Example 3, and loaded with in each case 4 ~l of an Ni2+
solution (O.lM NiS04/0.2M CH3-COONa, flow rate 5 ~l/min) of the given concentrations. After loading with the nickel had taken place, protein was injected and the resonance at t=l was determined. The plot of the resonance measured X1~61~
against the concentration of the nickel ion solution (Fig.
5) shows that complete loading of the surface is achieved using 4 ~l of a 100 ~M ~olution of Ni2+. Lower concentrations only result in incomplete loading of the surface, the extent of which depends on the concentration employed.
Example 5 The ability of different proteins to bind to the N-(5-amino-1-carboxypentyl) iminodiacetic acid surface In order to investigate the differing binding behaviour of different proteins, volumes of 40 ~l each of solutions (10 ~g/ml in running buffer) of a chick protein having the sequence depicted in the sequence listing, which sequence was modified with His(6) ((His)6-SCF), of bovine serum albumin (Sigma) and of egg white lysozyme (Sigma) were injected onto a biosensor surface which had been synthesized in accordance with Example 2. (The chick protein was prepared and purified following its expre~ion in a commercially available expression vector [pQE50, Diagen GmbH] in accordance with the purification protocol provided by the system manufacturer [The QIAexpressionist, Diagen GmbH, protocol 3, p. 35 and protocol 7, p. 45]. For its further purification, the protein was purified using an HQ/M anion exchange column [Perseptive Biosystems, Freiburg], and the protein concentration was determined by means of the Bradford protein assay [BioRad]). The binding behaviour of the individual proteins i8 depicted in Fig. 6: despite their relatively high concentrations, lysozyme (Mr 14300) and bovine serum albumin (Mr 68000) exhibit low resonance; by contrast, the His(6)-modified chick protein (Mr 22000) binds many times more strongly to the surface. It can also be seen that, in contrast to the His(6)-modified chick protein, lysozyme and bovine serum albumin achieve saturation of the surface. The surface which has been 19~15 loaded with the His (6) -modified chick protein to the point of saturation can be used to search for proteins, for example from the supernatants of chick cell line cell cultures, which interact with this protein by injecting cell culture supernatants from different cell lines. The binding of an interacting partner can be detected by the ampli~ication of the resonance which occurs following the injection.
Example 6 sinding of (His) 6-SCF to an N~ -di(1-carboxyethyl)-2,6-diaminohexanoic acid surface An N~,Na-di(1-carboxyethyl)-2,6-diaminohexanoic acid surface was prepared under exactly the same conditions as those described in Example 2 for N-(5-amino-1-carboxypentyl)iminodiacetic acid.
In order to investigate the behaviour of an (His) 6-modified protein when binding to this surface, the surface was loaded with Ni2+-ions, and 40 ~l of a solution (lo ~g/ml in running buf~er) o~ a chick protein having the His(6)-modified sequence ((His)6-SCF, described in Example 5) depicted in the sequence listing were injected onto this surface at a flow rate of 5 ~1/min. The sensorgram is depicted in Fig. 7. The surface shows a binding behaviour which is comparable to that of the surface which is modified with N-(5-amino-1-carboxypentyl)iminodiacetic acid and which is described in Example 2. The reproducibility of the surface immobilisation was confirmed by means of serial experiments using a ~tandardized solution of the protein.
- ~ ~19~
References Cuatrecasas, P. and Parikh, 1972, ~. Biochemi~try 11, 2291.
Hochuli, E., H. Dobel and Schacher, A., 1987, J.
Chromatography 411, 177-184.
Hochuli, E. and Piesecki, S., 1992, Methods 4 (San Diego), 66-72.
Lofas, S. and ~ohnsson, B., 1990, J. Chem. Soc., Chem.
Communications 1526.
Stenberg, E. et al., 1991, L. o~ Colloid and Interface Science, 143, 513.
Yokoyama, T., Sigeko, A. and Masatoshi, K., 1993, Chem.
Lett. 2, 383-386.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT
(A) NAME: Boehringer Ingelheim International GmbH
(B) STREET: Binger Strasse 173 (C) CITY: Ingelheim am Rhein (E) COUNTRY: FRG
(F) POSTAL CODE: 55216 (G) TELEPHONE: 06132/772282 (h) TELEFAX: 06132/774377 (ii) TITLE OF APPLICATION: Method for inve~tigating the interaction of biomolecules using surface plasmon resonance (iii) NUMBER OF SEQUENCES: 1 (iv) COMPUTER-READA;3LE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 204 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: no (vi) ORIGINAL SOURCE:
(A) ORGANISM: Gallus domesticus (G) CELL TYPE: fibroblast (ix) FEATURES:
(A) NAME/KEY: protein (B) LOCATION: 1..204 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
~ ~9~ 5 Met Ala Ser His His His Hi8 Hi8 His Gly Gly Ser Ala Gln Ser Ser Cys Gly Asn Pro Val Thr Asp Asp Val Asn Asp Ile Ala Lys Leu Val Gly Asn Leu Pro Asn Asp Tyr Leu Ile Thr Leu Lys Tyr Val Pro Lys Met Asp Ser Leu Pro Asn His Cys Trp Leu His Leu Met Val Pro Asp Phe Ser Arg Ser Leu His Asn Leu Leu Gln Lys Phe Ser Asp Ile Ser Asp Met Ser Asp Val Leu Ser Asn Tyr Ser Ile Ile Asn Asn Leu Thr Arg Ile Ile Asn Asp Leu Met Ala Cys Leu Ala Phe Asp Lys Asn Lys Asp Phe Ile Lys Glu Asn Gly His Leu Tyr Glu Glu Asp Arg Phe Ile Pro Glu Asn Phe Phe Ser Leu Phe Asn Ser Thr Ile Glu Val Tyr Lys Glu Phe Ala Asp Ser Leu Asp Lys Asn Asp Cys Ile Met Pro Ser Thr Val Glu Thr Pro Glu Asn Asp Ser Arg Val Ala Val Thr Lys Thr Ile Ser Phe Pro Pro Val Ala Ala Ser Ser Leu Arg Asn Asp Ser Ile Gly Ser Asn Thr Ser Ser Asn Ser Asn Lys Glu Ala Leu
Claims (26)
1. Method for investigating the interaction of a (poly)peptide with a reaction partner using a biosensor unit in which the surface plasmon resonance which is elicited by the interaction is determined in a metallic layer at the interface of two media which are permeable to electromagnetic radiation and which are of differing refractive index, with the medium of lower refractive index being an aqueous medium in which the (poly)peptide is present in immobilized form and is brought into contact with the reaction partner, characterized in that the (poly)peptide is immobilized by way of a metal chelate.
2. Method according to Claim 1, characterized in that the aqueous medium is a biocompatible, porous matrix, in particular a hydrogel.
3. Method according to Claim 2, characterized in that the chelating agent is bound to the surface of the biosensor unit by way of reactive groups possessed by the hydrogel.
4. Method according to Claim 2 or 3, characterized in that the hydrogel is selected from the group comprising polysaccharides such as dextran, agarose, carrageen, alginates, starch, cellulose or derivatives thereof, or water-swellable organic polymers such as polyvinyl alcohol, polyacrylic acid, polyacrylamide or polyethyleneglycol.
5. Method according to Claim 4, characterised in that the hydrogel is a dextran.
6. Method according to Claim 5, characterized in that the dextran possesses carboxymethyl groups as reactive groups.
7. Method according to one of the preceding claims, characterized in that the (poly)peptide is a fusion (poly)peptide which, in addition to its biologically active segment, possesses an affinity peptide having at least one histidine residue, and the chelating agent is an iminodiacetic acid derivative.
8. Method according to Claim 7, characterized in that the (poly)peptide possesses an affinity peptide having at least two adjacent histidine residues, and the chelating agent is a nitrilotriacetic acid derivative of the general formula Y-R-CH(COOH)-N(CH2COOH)2, wherein R may denote an alkylene group of the type (CH2)n- which may be substituted or unsubstituted, with the proviso that the substituent does not have a detrimental effect on the function of the chelating agent, and n denotes an integer 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, whilst if the alkylene group is large enough it may possibly also contain one or more alkene or alkyne partial structures, or wherein R may denote an aromatic bridge member which may be made up of one or more mono- or polynuclear aromatic compounds which may optionally also be an aromatic heterocycle, or wherein R may denote an aralkyl bridge member in which the aromatic moiety may be bound to Y or to the .alpha.-C
atom adjacent to the carboxyl group, either directly or via an alkyl group of type (CH2)n-, wherein n may denote an integer 1, 2, 3, 4 or 5, and wherein Y is a reactive group, particularly an NH2 or SH group.
atom adjacent to the carboxyl group, either directly or via an alkyl group of type (CH2)n-, wherein n may denote an integer 1, 2, 3, 4 or 5, and wherein Y is a reactive group, particularly an NH2 or SH group.
9. Method according to Claim 8, characterized in that the nitrilotriacetic acid derivative is N-(5-amino-1-carboxypentyl)iminodiacetic acid.
10. Method according to Claim 7, characterized in that the (poly)peptide possesses an affinity peptide having at least two adjacent histidine residues, and the chelating agent is a nitrilotriacetic acid derivative of the general formula Y-R1-CH(COOH)-N(R2CHCOOH)2, in which R1 is a group having the meanings given for R in claim 8, and wherein R2 may be an alkyl group of the type CH3(CH2)n- which may be substituted, e.g. with OH or Cl, or unsubstituted, and n may denote an integer 1, 2, 3, 4 or 5, or wherein R2 may be a branched alkyl group such as isopropyl, t-butyl or isobutyl, or wherein R2 may be an aromatic bridge member having the meaning defined for R in claim 8, and wherein Y is a reactive group, particularly an NH2 or SH group.
11. Method according to Claim 10, characterized in that the nitrilotriacetic acid derivative is N.alpha.,N.alpha.-di(1-carboxyethyl)-2,6-diaminohexanoic acid.
12. Method according to one of Claims 7 to 11, characterized in that the chelating agent is complexed with a transition metal ion, particularly of the fourth period.
13. Process according to one of claims 7 to 12, characterised in that the chelating agent is complexed with Ni2+ ions.
14. Nitrilotriacetic acid derivative of the formula N.alpha.,N.alpha.-di(1-carboxyethyl)-2,6-diaminohexanoic acid.
15. Use of the nitrilotriacetic acid derivative according to claim 14 for purifying proteins and peptides by metal chelate affinity chromatography.
16. Biosensor unit for investigating the interaction of a (poly)peptide with a reaction partner by means of determining the surface plasmon resonance in a metallic layer at the interface of two media which are permeable to electromagnetic radiation and which are of differing refractive index, with the medium of lower refractive index being an aqueous medium, characterized in that a chelating agent, where appropriate in a form in which it is complexed with a metal ion, is bound to the surface of the biosensor unit which faces the aqueous medium.
17. Biosensor unit according to Claim 16, characterized in that the chelating agent is bound to the reactive groups of a biocompatible, porous matrix, in particular a hydrogel.
18. Biosensor unit according to claim 17, characterised in that the hydrogel is selected from the group comprising polysaccharides such as dextran, agarose, carageen, alginates, starch, cellulose or derivatives thereof, or water-swellable organic polymers such as polyvinyl alcohol, polyacrylic acid, polyacrylamide or polyethyleneglycol.
19. Biosensor unit according to Claim 18, characterized in that the hydrogel is a dextran.
20. Biosensor unit according to Claim 19, characterized in that the dextran possesses carboxymethyl groups as reactive groups.
21. Biosensor unit according to one of Claims 16 to 20, characterized in that the chelating agent is an iminodiacetic acid derivative.
22. Biosensor unit according to Claim 21, characterized in that the chelating agent is a nitrilotriacetic acid derivative of the general formula Y-R-CH(COOH)-N(CH2COOH)2, wherein R may denote an alkylene group of the type (CH2)n- which may be substituted or unsubstituted, with the proviso that the substituent does not have a detrimental effect on the function of the chelating agent, and n denotes an integer 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, whilst if the alkylene group is large enough it may possibly also contain one or more alkene or alkyne partial structures, or wherein R may denote an aromatic bridge member which may be made up of one or more mono- or polynuclear aromatic compounds which may optionally also be an aromatic heterocycle, or wherein R may denote an aralkyl bridge member in which the aromatic moiety may be bound to Y or to the .alpha.-C
atom adjacent to the carboxyl group, either directly or via an alkyl group of type (CH2)n-, wherein n may denote an integer 1, 2, 3, 4 or 5, and wherein Y is a reactive group, particularly an NH2 or SH group.
atom adjacent to the carboxyl group, either directly or via an alkyl group of type (CH2)n-, wherein n may denote an integer 1, 2, 3, 4 or 5, and wherein Y is a reactive group, particularly an NH2 or SH group.
23. Biosensor unit according to Claim 22, characterized in that the nitrilotriacetic acid derivative is N-(5-amino-1-carboxypentyl)iminodiacetic acid.
24. Biosensor unit according to Claim 21, characterized in that the (poly)peptide possesses an affinity peptide having at least two adjacent histidine residues, and the chelating agent is a nitrilotriacetic acid derivative of the general formula Y-R1-CH(COOH)-N(R2CHCOOH)2, in which R1 is a group having the meanings given for R in claim 8, and wherein R2 may be an alkyl group of the type CH3(CH2)n- which may be substituted or unsubstituted, and n may denote an integer 1, 2, 3, 4 or 5, or wherein R2 may be a branched alkyl group such as isopropyl, t-butyl or isobutyl, or wherein R2 may be an aromatic bridge member having the meaning defined for R in claim 8, and wherein Y is a reactive group, particularly an NH2 or SH group.
25. Biosensor unit according to Claim 24, characterized in that the nitrilotriacetic acid derivative is N.alpha.,N.alpha.-di(1-carboxyethyl)-2,6-diaminohexanoic acid.
26. Biosensor kit for investigating the interaction of a (poly)peptide with a reaction partner using surface plasmon resonance (SPR), which kit contains an SPR biosensor unit in a first container, a chelating agent in a further container, a salt of a metal which is suitable for complexing with the chelating agent in a further container, the reagents for activating the surface of the biosensor unit in one or more additional containers, where appropriate a reagent for deactivating the surface in another container, where appropriate a reagent for regenerating the surface in another container, and also, where appropriate, one or more comparison proteins in one or more additional containers.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4433980.1 | 1994-09-23 | ||
| DE4433980A DE4433980C2 (en) | 1994-09-23 | 1994-09-23 | Process and biosensor hit for investigating the interaction of biomolecules by means of surface plasma resonance |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2198615A1 true CA2198615A1 (en) | 1996-03-28 |
Family
ID=6528988
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
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| CA002198615A Abandoned CA2198615A1 (en) | 1994-09-23 | 1995-09-21 | Method of investigating the interaction of biomolecules using surface plasmon resonance |
Country Status (12)
| Country | Link |
|---|---|
| EP (1) | EP0784792A2 (en) |
| JP (1) | JPH10505910A (en) |
| KR (1) | KR970706494A (en) |
| CN (1) | CN1158658A (en) |
| AU (1) | AU3608995A (en) |
| BR (1) | BR9509077A (en) |
| CA (1) | CA2198615A1 (en) |
| CO (1) | CO4410395A1 (en) |
| DE (1) | DE4433980C2 (en) |
| PL (1) | PL319354A1 (en) |
| WO (1) | WO1996009547A2 (en) |
| ZA (1) | ZA958024B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6165335A (en) | 1996-04-25 | 2000-12-26 | Pence And Mcgill University | Biosensor device and method |
| US6107080A (en) * | 1996-04-25 | 2000-08-22 | Mcgill University | Biosensor device and method |
| US6130037A (en) * | 1996-04-25 | 2000-10-10 | Pence And Mcgill University | Biosensor device and method |
| US5955379A (en) * | 1996-04-25 | 1999-09-21 | Mcgill University | Biosensor device and method |
| AU6883198A (en) * | 1997-04-24 | 1998-11-13 | American Home Products Corporation | Method for the identification and characterization of nuclear receptor ligands |
| CA2342767C (en) * | 1998-09-03 | 2009-11-03 | Trellis Bioinformatics, Inc. | Multihued labels |
| AUPP856399A0 (en) * | 1999-02-08 | 1999-03-04 | Australian Membrane And Biotechnology Research Institute | Improved compounds for protein binding |
| US6787368B1 (en) | 1999-03-02 | 2004-09-07 | Helix Biopharma Corporation | Biosensor method for detecting analytes in a liquid |
| US7804592B2 (en) | 2003-10-16 | 2010-09-28 | Nard Institute, Ltd. | Method for measuring a surface plasmon resonance and noble metal compound used for the same |
| EP1674857B1 (en) * | 2003-10-16 | 2014-04-02 | Kabushiki Kaisha Nard Kenkyusho | Method for measuring a surface plasmon resonance and noble metal compound used for the same |
| CN101261226B (en) * | 2007-03-08 | 2010-12-08 | 北京宏荣博曼生物科技有限责任公司 | Surface plasma resonance instrument chip based on polyethyleneglycol |
| JP2010197046A (en) * | 2007-05-28 | 2010-09-09 | Tanaka Holdings Kk | Biosensor |
| EP2015071A1 (en) | 2007-07-13 | 2009-01-14 | FUJIFILM Corporation | Carrier, process for producing same, bioreactor, and chip for surface plasmon resonance analysis |
| KR100953558B1 (en) | 2007-12-12 | 2010-04-21 | 한국전자통신연구원 | PS-SPCL searching apparatus and method using surface plasmon resonance |
| CN114797804B (en) * | 2022-03-29 | 2023-08-04 | 翌圣生物科技(上海)股份有限公司 | NTA chromatographic medium with long connecting arm and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4569794A (en) * | 1984-12-05 | 1986-02-11 | Eli Lilly And Company | Process for purifying proteins and compounds useful in such process |
| CA1304886C (en) * | 1986-07-10 | 1992-07-07 | Heinz Dobeli | Metal chelate resins |
| IL85137A (en) * | 1987-01-21 | 1992-02-16 | Ares Serono Res & Dev Ltd | Method of assaying for a ligand using surface plasmon resonance effect |
| CA1340522C (en) * | 1987-03-10 | 1999-05-04 | Heinz Dobeli | Fusion proteins containing neighbouring histidines for improved purification |
| DE58905660D1 (en) * | 1988-04-25 | 1993-10-28 | Hoffmann La Roche | Diagnostic tools. |
| SE462454B (en) * | 1988-11-10 | 1990-06-25 | Pharmacia Ab | METHOD FOR USE IN BIOSENSORS |
| US5169936A (en) * | 1989-04-14 | 1992-12-08 | Biogen, Inc. | Protein purification on immobilized metal affinity resins effected by elution using a weak ligand |
| DE59106527D1 (en) * | 1990-11-14 | 1995-10-26 | Hoffmann La Roche | Immunosensitive transducer and method for its production. |
-
1994
- 1994-09-23 DE DE4433980A patent/DE4433980C2/en not_active Expired - Fee Related
-
1995
- 1995-09-21 CN CN95195182A patent/CN1158658A/en active Pending
- 1995-09-21 CA CA002198615A patent/CA2198615A1/en not_active Abandoned
- 1995-09-21 CO CO95043762A patent/CO4410395A1/en unknown
- 1995-09-21 PL PL95319354A patent/PL319354A1/en unknown
- 1995-09-21 EP EP95933414A patent/EP0784792A2/en not_active Ceased
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- 1995-09-21 AU AU36089/95A patent/AU3608995A/en not_active Abandoned
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Also Published As
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| DE4433980A1 (en) | 1996-03-28 |
| ZA958024B (en) | 1996-03-25 |
| CN1158658A (en) | 1997-09-03 |
| WO1996009547A2 (en) | 1996-03-28 |
| JPH10505910A (en) | 1998-06-09 |
| DE4433980C2 (en) | 1996-08-22 |
| KR970706494A (en) | 1997-11-03 |
| WO1996009547A3 (en) | 1996-05-30 |
| PL319354A1 (en) | 1997-08-04 |
| EP0784792A2 (en) | 1997-07-23 |
| CO4410395A1 (en) | 1997-01-09 |
| AU3608995A (en) | 1996-04-09 |
| BR9509077A (en) | 1997-11-25 |
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