JP4135791B2 - Biomaterial purification method using electrocleavable affinity carrier, electrocleavable affinity carrier, biomaterial purification kit, electrocleavable linker - Google Patents

Biomaterial purification method using electrocleavable affinity carrier, electrocleavable affinity carrier, biomaterial purification kit, electrocleavable linker Download PDF

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JP4135791B2
JP4135791B2 JP2004530574A JP2004530574A JP4135791B2 JP 4135791 B2 JP4135791 B2 JP 4135791B2 JP 2004530574 A JP2004530574 A JP 2004530574A JP 2004530574 A JP2004530574 A JP 2004530574A JP 4135791 B2 JP4135791 B2 JP 4135791B2
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carrier
electrocleavable
linker
electrolytic
ligand
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JPWO2004018081A1 (en
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一裕 千葉
信弘 高橋
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農工大ティー・エル・オー株式会社
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    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D495/00Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04Ortho-condensed systems

Description

  The present invention relates to a method for purifying biological material and an electrocleavable affinity carrier for use in the method. The present invention also relates to a biological material purification kit using an electrocleavable affinity carrier, and an electrocleavable linker.

Various methods are known for separating only a target protein from a solution containing biological substances such as contaminating proteins. For example, a method in which a target protein is expressed as a tagged fusion protein by a recombinant technique and only a tagged protein is selectively bound using a ligand specific to the tag is widely used. In this method, in order to recover the target protein, the salt concentration, pH, or solvent composition is changed, or after selective binding, the tag portion is cleaved with a highly specific protease and only the target protein is recovered. the eluting (e.g., "a generic protein purification method for protein complex characterization and proteome exploration", Guillaume Rigaut, Anna Shevchenko, Berthold Rutz, Matthias Wilm, Matthias Mann, Bertrand Seraphin, Nature Biotechnology17,1030-1032 (01 Oct 1999) ). However, in this method, nonspecifically adsorbed contaminant proteins may be eluted. In addition, the method of cleaving with a protease has a drawback that the target protein itself is cleaved if the protease recognition site is present in the target protein.
In addition, a method of recovering a protein using a synthetic peptide corresponding to the antigen determination site of the protein after binding a biological substance such as a protein using an antibody against the target protein as a ligand, This method has low protein recovery efficiency. In particular, when the antigen determining site is unknown, it is not possible to selectively elute only the target protein, and the recovery rate of the biological material containing the protein is further reduced.
A method has also been developed in which a ligand is bound to a carrier via a light-sensitive linker, a protein is bound to the ligand, and then the linker is cleaved by applying light. In this method, when the carrier is packed in the column, the sample cannot be sufficiently irradiated with light in the column. In addition, when the light energy is weak, sufficient cleavage cannot be obtained, but when the light energy is too strong, the structure of the target protein may change, and it is difficult to adjust the light intensity.
In addition, cysteine residues, phosphorylated serine, threonine, and tyrosine residues of the target protein are modified with a biotin-linked modifying reagent, and cysteine, phosphorylated serine, A method for recovering only a peptide containing a threonine / tyrosine residue has also been proposed, but it has the disadvantage of a very low recovery rate.
A method is also known in which a drug is immobilized on a carrier as an affinity ligand, a target protein of the target drug is bound, and the target protein is recovered using the drug. In particular, hydrophobic drugs have low solubility in aqueous solutions and low protein recovery. In addition, DNA fragments, RNA fragments, or biological substances other than proteins are used as ligands, biological substances such as proteins are bound, and the original structure of biological substances is destroyed using urea or surfactants. Although a method for recovering a biological material is also known, contaminants are also recovered at the same time, and the target biological material cannot be selectively recovered at a high recovery rate. These problems are the same not only for proteins but also for other biological macromolecules and biologically active substances such as physiologically active substances.
Therefore, an object of the present invention is to provide a biological material purification method capable of obtaining a target biological material with a high recovery rate by simple means, and an affinity carrier for use in such a method.

The present inventors have found that the target biological material can be easily recovered by binding the biological material to the affinity carrier and then cleaving the affinity carrier by an electrochemical method, thereby completing the present invention. It was.
That is, the present invention is a method for purifying a biological material, wherein an aqueous solution containing a biological material such as a protein is brought into contact with an electrocleavable affinity carrier, and a voltage is applied to the aqueous solution to apply the electrocleavable affinity. There is provided a method comprising recovering a biological substance such as the protein by cleaving a carrier.
The present invention can be applied to all biological materials separated and purified by affinity chromatography technology using affinity (affinity by affinity). As used herein, biological material includes both biological macromolecules and biological small molecules. Examples of the former include proteins, nucleic acids (DNA, RNA), enzymes, sugars, sugar chains, etc., and examples of the latter include peptides, lipids, vitamins, metal complexes, steroids, terpenoids (terpenes), otachoids, alkaloids, etc. Of physiologically active substances.
The electrolytically cleavable affinity carrier is a carrier for use in affinity purification and has a characteristic that a part thereof is cleaved by applying a voltage. Preferably, the electrocleavable affinity carrier is composed of a ligand bound to a solid carrier via an electrocleavable linker. An electrolytically cleavable linker is a linker that can be cleaved by applying a voltage. Preferably, the electrocleavable linker comprises an electrocleavable moiety, at least one ligand binding arm coupled to the electrolytic cleavage moiety, and at least one carrier binding arm coupled to the electrolytic cleavage moiety. Particularly preferably, the carrier binding arm is bound to the carrier by a platinum oxide-silicon bond.
In another aspect, the present invention provides an electrocleavable affinity carrier comprising a ligand bound to a solid carrier via an electrocleavable linker. A ligand refers to a substance that can selectively or specifically bind to a target substance to be purified. Examples of ligands include, but are not limited to, antigens, antibodies, haptens, peptides, receptors, binding partners that bind to receptors, biotin, avidin, protein A, and the like. The electrocleavable affinity carrier of the present invention is characterized by being cleaved by applying a voltage. Preferably, the electrocleavable linker comprises an electrocleavable moiety, at least one ligand binding arm coupled to the electrolytic cleavage moiety, and at least one carrier binding arm coupled to the electrolytic cleavage moiety. Particularly preferably, the carrier binding arm is bound to the carrier by a platinum oxide-silicon bond.
In still another aspect, the present invention provides a kit for purifying biological materials such as a protein containing the above-described electrocleavable affinity carrier.
In another aspect, the present invention provides an electrocleavable linker that is cleaved by applying a voltage, the electrolysis cleavage portion, at least one ligand-binding arm bound to the electrolysis cleavage portion, and the electrolysis cleavage. An electrocleavable linker is provided that includes at least one carrier binding arm attached to the moiety. At least one carrier can be bound to the electrocleavable linker via a carrier binding arm.
By using the electrocleavable affinity carrier of the present invention, after binding the target biological substance to the ligand, a weak voltage is applied between the carrier and the aqueous solution, so that the electrocleavable affinity carrier is a linker therein. Since it is cut | disconnected in this specific site | part, biological material can be collect | recovered efficiently. Since the biological material purification method according to the present invention has a high biological material recovery rate, it is particularly useful when recovering a plurality of biological materials whose structures are unknown from a very small amount of sample.

FIG. 1 shows one embodiment of the constitution of the electrocleavable affinity carrier of the present invention.
FIG. 2 shows a schematic diagram of a column packed with the electrocleavable affinity carrier of the present invention.
FIG. 3 shows a preferred example of the structure of the electrocleavable linker of the present invention.
FIG. 4 shows a preferred example of the structure of the electrocleavable linker of the present invention.
FIG. 5 shows a preferred example of the structure of the electrocleavable linker of the present invention.
FIG. 6 shows a preferred example of the structure of the electrocleavable linker of the present invention.
FIG. 7 shows a cross-sectional view of a BIAcore chip and a schematic diagram of the principle for detecting the interaction between a biological substance and a ligand using the BIAcore chip.
FIG. 8 is a schematic diagram in which the electrocleavable affinity carrier of the present invention is applied to a BIAcore chip.
FIG. 9 shows a preferred example of the electrocleavable linker of the present invention bonded to a carrier by a platinum oxide-silicon bond.
FIG. 10 shows an example of electrolytic cleavage using the electrolytically cleavable affinity carrier of the present invention.
FIG. 11 shows an example of an apparatus suitable for the production and use of the electrocleavable affinity carrier of the present invention.
FIG. 12 shows the electrocleavable affinity carrier of the present invention using biotin as a ligand.
FIG. 13 shows a method for producing an electrocleavable affinity carrier of the present invention using biotin as a ligand.
FIG. 14 shows the binding and cleavage of avidin using the electrocleavable affinity carrier of the present invention using biotin as the ligand.
DETAILED DESCRIPTION OF THE INVENTION FIG. 1 shows one embodiment of the construction of the electrocleavable affinity carrier of the present invention. In this embodiment, the electrocleavable affinity carrier of the present invention is composed of a ligand bound to a solid carrier via an electrocleavable linker. Preferably, the electrocleavable linker comprises an electrocleavable moiety, at least one ligand binding arm coupled to the electrolytic cleavage moiety, and at least one carrier binding arm coupled to the electrolytic cleavage moiety.
As the carrier, any metal can be used as long as it has a lower ionization tendency than hydrogen, but gold and platinum are preferred. The reason is that, since the affinity between gold and platinum and the biological material is low, impurities are not easily mixed in, and it is difficult to change and can be reused. Of course, another metal or other conductive material with gold or platinum deposited or plated may be used. Carbon (carbon black or the like) can also be used as the carrier. In this case, it is preferable to prevent the target protein from being adsorbed on the carrier instead of the ligand by derivatizing the surface of the carbon carrier with a carboxyl group.
The carrier can be in the same shape as a carrier generally used for protein or biological substance affinity purification in the technical field, for example, a bead shape having a diameter of 0.1 μm-1 mm. Preferably, beads with a diameter of 1-20 μm are used to increase the surface area in contact with the protein to be purified. Alternatively, the carrier may be in the form of a sponge or porous matrix.
The electrolytic cleavage portion is an aromatic group that is cleaved along with electron transfer when a voltage is applied. For example, the following groups can be used:
[Wherein X is a carbon, oxygen, nitrogen or sulfur atom, R is a hydrogen, hydrocarbon group, acyl group or a substituent bonded via carbon, oxygen, nitrogen or sulfur, and ■ is a ligand bond Arm or carrier binding arm].
Preferred examples of the electrolytic cleavage moiety that can be used in the present invention include, but are not limited to, the following groups. A suitable electrolytic cleavage moiety that can be used in the present invention can be easily designed and synthesized based on the disclosure of the present specification, as long as the person is familiar with technical fields such as affinity purification.
The ligand binding arm is a spacer group for binding the electrocleavable moiety and the ligand at an appropriate distance. The ligand binding arm is preferably an alkylene chain having a length of 3 to 100 carbon atoms, wherein one or more non-adjacent —CH 2 — groups are independently —NH—, —NHCO. It may be substituted with-, -CO-, -O- or -O-CO-. The ligand binding arm has a functional group for binding a ligand at the terminal end, such as a —COOH group or —NH 2 group, or a succinimide group or an epoxy group. In addition, a reactive functional group of any compound used for protein modification, amino acid modification, nucleic acid modification, and other biological substance modification may be added to the end of the ligand binding arm. Preferred examples of ligand binding arms that can be used in the present invention include, but are not limited to, the following groups:
One skilled in the art can readily design and synthesize appropriate ligand binding arms that can be used in the present invention based on the disclosure herein. In particular, the length of the ligand-binding arm and the hydrophobicity / hydrophilicity based on the physical properties (eg, molecular size, charge) and chemical properties of the biological material such as the ligand used for affinity purification and the protein to be purified. Can be appropriately selected.
The carrier binding arm is a spacer group for binding the electrocleavable moiety and the carrier at an appropriate distance. The carrier binding arm is preferably shorter than the ligand binding arm (3-100 carbons in length), more preferably an alkylene chain with 3-10 carbons in length, where 1 or The above —CH 2 — groups may be independently substituted with —NH—, —NHCO—, —CO—, —O— or —O—CO—. If the distance between the electrolytically cleavable part and the carrier is a certain length (determined by the voltage, the properties of the electrolyte solution, the properties of the electrolytically cleavable part, etc.), the electrolytically cleavable affinity carrier of the present invention is When a voltage is applied, it is cut at the electrolytic cutting portion. When the length of the carrier binding arm is longer than this, the electrocleavable affinity carrier of the present invention is moved, so that the cleavage occurs when a certain distance or less is reached from the carrier. Therefore, a particularly preferable length of the carrier binding arm is 3 to 6 carbon atoms. In the case where the length of the carrier-binding arm is longer, such a mode is also preferable since various properties such as hydrophilicity and hydrophobicity can be adjusted by incorporating various functional groups into the arm. The carrier binding arm has a functional group for binding to the carrier at the end. When the support is gold, a suitable functional group is a —SH group, and when the support is platinum, a suitable functional group is —Si (EtO) 3 or —Si (MeO) 3 . Alternatively, when the surface of the support is derivatized with —COOH, —NH 2 groups, etc., it is a functional group capable of binding to these functional groups, for example, —OH, —NH 2 , —COOH, etc. Also good. Preferred examples of the carrier binding arm that can be used in the present invention include, but are not limited to, the following groups.
One skilled in the art can readily design and synthesize suitable carrier binding arms that can be used in the present invention based on the disclosure herein. In particular, the length of the carrier binding arm and the hydrophobicity or hydrophilicity can be appropriately selected based on the properties of the biological substance such as the protein to be purified and the material of the carrier.
In a particularly preferred embodiment of the present invention, the carrier and the carrier binding arm are bound by a platinum oxide-silicon bond. When the linker is cleaved by electrolytic oxidation using the carrier as an anode, if the carrier and the carrier binding arm are bonded by, for example, an Au-S bond, this bonded portion may also be oxidatively cleaved. When cleavage occurs between the carrier and the electrolytically cleavable linker, the proportion of nonspecifically bound contaminant components increases at the same time, so it is necessary to carefully set the conditions for electrolytic cleavage. On the other hand, since the platinum oxide-silicon bond is hardly cleaved by the oxidation treatment in an aqueous solution, it is possible to cause an electrolytic oxidative cleavage reaction selectively at the electrolytically cleavable portion. This makes it easy to control the reaction and has the advantage that it can be selectively disconnected by an external switch without being affected by the reduction reaction system contained in the mixture of biological components.
FIGS. 3-6 show preferred examples of the structure of the electrocleavable linker of the present invention. The scope of the present invention is not limited to these.
By binding at least one ligand to the electrocleavable linker of the present invention via a ligand binding arm and further binding at least one carrier via a carrier binding arm, an electrocleavable affinity carrier can be obtained. . The electrocleavable affinity carrier of the present invention is useful for purifying biological substances such as proteins with a high recovery rate.
The structure of the electrocleavable affinity carrier of the present invention is shown in FIG. The electrolytically cleavable affinity carrier of the present invention is brought into contact with an aqueous solution containing a biological substance such as a target protein and washed to remove biological substances such as contaminating proteins. At this time, some of the contaminants may remain adsorbed nonspecifically with the carrier. Next, by applying a voltage between the carrier and the aqueous solution, the electrocleavable portion of the electrocleavable affinity carrier is cleaved. The voltage to be applied is 0.5V-1.5V, more preferably 0.5V-1.2V, and most preferably 1V. The lower the voltage, the lower the cutting efficiency. When the voltage is higher than 1.5V, water is electrolyzed.
As a method for applying the voltage, in addition to the application of a constant voltage, any method such as a pulse voltage application and a method of applying a reciprocal sweep (sweep) with a sine wave, a sawtooth wave, a triangular wave or the like can be adopted. When the electrolytic cutting portion is cut at a single specific voltage, a constant voltage higher than that voltage may be applied continuously. However, since there are many variations in the cutting voltage, it is preferable that the voltage range that covers the maximum and minimum cutting voltages be swept and applied more reliably.
FIG. 2 shows an embodiment of protein purification using a column packed with the electrocleavable affinity carrier of the present invention. The electrolytically cleavable affinity carrier of the present invention is packed in a column together with an electrolyte solution, and an electrode is mounted so as to be in electrical communication with the carrier. The counter electrode is mounted in electrical communication with the electrolyte solution. The solution containing the protein to be purified is loaded onto the column and the protein is bound to the ligand. When the column is washed to remove contaminating proteins and then a voltage is applied to the electrode, the electrocleavable linker is cleaved at the electrocleavable portion, and the protein bound to the ligand is dissociated from the carrier. By eluting the dissociated protein from the column, the target protein can be recovered.
FIG. 11 shows an example of an apparatus suitable for the production and use of the electrocleavable affinity carrier of the present invention. FIG. 11A is an example of a reaction column that can be used to attach the electrocleavable linker of the present invention to a platinum support. When carrying out the electrolytic reaction, electrodes are mounted as shown in FIG. 11B. Such a column is attached to the suction bottle shown in FIG. 11C, and a reaction solution, a washing solution or an eluate is added from above. The solution can be recovered inside the bottle by aspirating the system after contacting the solution and platinum particles for a desired time.
By using the electrocleavable affinity carrier of the present invention, by appropriately selecting the conditions for eluting the protein, it is possible to prevent elution of contaminating proteins adsorbed non-specifically on the carrier binding arm of the carrier or the electrocleavable linker. Therefore, there is an advantage that contamination protein is very little mixed in the recovered protein.
The present invention also provides a biological material purification kit comprising the electrocleavable affinity carrier of the present invention. The electrocleavable affinity carrier may be provided in a form packed in a column. The kit may further include a solution for dissolving the sample, a washing solution, and an eluent. Alternatively, the kit may include an electrocleavable linker bound to a carrier, in which case the user can bind a ligand to the linker depending on the purpose.
The electrocleavable affinity carrier of the present invention is particularly easily applicable to a biochip for real-time BIA (Biomolecular Interaction Analysis). The biochip for BIA is a trade name of BIAcore (registered trademark; hereinafter referred to as “BIAcore chip”), and is commercially available from Biacore AB (Uppsala, Sweden). Such chip technology is disclosed in USP 5,641,640, USP 5,955,729, Japanese Patent No. 3294605, and Japanese Translation of PCT National Publication No. 07-507865, and the outline thereof will be described below.
The BIAcore chip uses a surface plasmon resonance (SPR) phenomenon to detect an interaction between molecules on the surface of the sensor chip. In order to allow biological substances such as ligands and proteins to interact, a fine flow channel through which these substances flow is formed on the surface of the BIAcore chip. Immobilize the ligand on its surface. FIG. 7 shows a cross-sectional view of a BIAcore chip and a schematic diagram of the principle for detecting the interaction between a biological substance and a ligand using the BIAcore chip.
In FIG. 7, F is a biological material flow channel, G is a gold deposition film, P is polarized light, and R is totally reflected light. L is a ligand, D is dextran, and DL is a dextran binding linker. On the surface of the BIAcore chip, dextran having a length of about 100 nm is bound, and the target ligand is bound to the dextran. As shown in the figure, a single ligand may be attached to a single dextran, or a ligand may be attached to a dextran in which a plurality of ligands are branched like a fruit. In SPR, it is said that detection is possible when a substance binds to a ligand within a distance of about 100 nm or less. When the substance and the ligand fixed on the surface interact with each other, a refractive index change due to SPR occurs, and this is optically measured on the back surface of the sensor chip. Thus, the interaction between the ligand and the biological substance containing the protein can be detected, and the interaction can be quantified from the reflection angle of the reflected light. As shown in FIG. 7, on the surface of the BIAcore chip, a gold vapor deposition film with good optical characteristics (high total reflectance) is used for using SPR.
Since the gold thin film is used, the electrocleavable affinity carrier of the present invention can be easily applied to the BIAcore chip. That is, the electrocleavable affinity carrier of the present invention can be bound to the gold thin film surface of the BIAcore chip. Alternatively, the electrocleavable affinity carrier of the present invention may be bound to a part of conventionally used dextran. Then, one of the electrodes for applying the voltage is electrically connected to the gold thin film of the BIAcore chip. It is easy to add such an electric circuit. In this way, the interaction between the substance and the fixed ligand can be monitored quantitatively in real time by the real-time BIA technology, and the fixed ligand can be separated at a desired timing. For example, if the binding between the ligand and the protein is detected, then the washing reagent is run in the flow of the chip surface, and the binding protein is separated after the washing is completed. can do.
FIG. 8 is a schematic view of an example in which the electrocleavable affinity carrier of the present invention is applied to a BIAcore chip. LA is a ligand binding arm, CP is a cleavage site, and CA is a carrier binding arm. The length of the carrier binding arm is selected so that the electrocleavable affinity carrier is cut at the cutting site when a voltage is applied using a gold vapor deposition film as an electrode. Alternatively, by selecting a longer carrier binding arm length, the electrocleavable affinity carrier of the present invention moves non-stationarily in the flow channel flow with the ligand immobilized on the surface of the BIAcore chip. It may be cut when the part moves a predetermined distance from the carrier.
The contents of all patents and references explicitly cited herein are hereby incorporated by reference as part of the present specification. In addition, the contents described in the specification and drawings of Japanese Patent Application No. 2002-241072, which is an application on which the priority of the present application is based, are cited herein as part of the present specification.

EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples. The following examples show chemical synthesis and cleavage of the electrocleavable linker of the present invention that cleaves upon electron transfer, and examples of protein purification using the same.
Example 1. Synthesis of linker having an electrolytic cleavage unit attached through a sulfur atom onto gold particles
p-Hydroxybenzaldehyde (10 mmol)
And 3-mercapto-propionic acid (22 mmol)
Was dissolved in dichloromethane at room temperature. Next, boron trifluoride-diethyl ether complex (0.1 mmol) was added and stirred for 5 minutes. Immediately, brine was added to the reaction solution, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed with brine. The ethyl acetate solution was dried over anhydrous sodium sulfate, filtered, concentrated to dryness, and purified by silica gel column chromatography (elution solvent hexane-ethyl acetate) to give 3-[(2-carboxy-ethylsulfanyl)-(4- Hydroxy-phenyl) -methylsulfanyl] -propionic acid was obtained.
To 3-mercapto-propionic acid (5 mmol), 20 mmol of thionyl chloride was newly added and stirred at room temperature for 1 hour. The solution was concentrated under reduced pressure, dichloromethane was added, and 3-[(2-carboxy-ethylsulfanyl)-(4-hydroxy-phenyl) -methylsulfanyl] -propionic acid (5 mmol) and triethylamine (20 mmol) prepared earlier were further added. ) And stirred at room temperature for 16 hours under a stream of argon. After completion of the reaction, 5% aqueous citric acid solution and ethyl acetate were added, and the product was extracted with ethyl acetate. The ethyl acetate layer was further washed with a 5% aqueous citric acid solution, dried over anhydrous sodium sulfate, filtered and concentrated. Acetonitrile was added to the residue for dissolution, gold particles were further added, and the mixture was allowed to stand at room temperature for 18 hours under a stream of argon. Subsequently, the gold particles were separated by filtration, washed 5 times with acetonitrile, and then immersed in dimethylformamide. Dicyclohexylcarbodiimide (10 mmol) and 2,2 '-(ethylenedioxy) bis (ethylamine) (20 mmol)
And stirred at room temperature for 4 hours. Next, the gold particles were filtered off and washed 5 times with acetonitrile. The gold particles thus obtained have an amino group at the terminal, and any ligand can be bound to the amine.
Example 2 Benzoylation was performed by reacting benzoyl chloride as a ligand model with an electrochemically cleaved terminal amine in aqueous solution . After the chemically modified gold particles were dispersed in dimethylformamide, an excess amount of benzoyl chloride was added and stirred at room temperature for 3 hours. After cooling the reaction vessel with ice, methanol was added dropwise. Thereafter, the gold particles were separated, washed 5 times with acetonitrile, and further washed 3 times with pure water. The obtained gold particles were packed in a column tube as shown in FIG. 2, and after setting a platinum lead, 50 mM phosphate buffer solution (pH 7.2) was filled. Next, with the gold particle side as the anode, the potential was repeatedly swept between the terminal voltage of 0 v and 1.6 V (reciprocating sweep, 100 mV / sec). After 40 sweeps, the lead was disconnected and the buffer was eluted from the column. From this buffer solution, the ligand unit cleaved at the dithioacetal moiety by electrolysis was recovered.
Example 3 Synthesis of linkers with electrolytic cleavage units bonded to platinum-platinum oxide particles via silicon atoms
2,5-Dihydroxybenzaldehyde (10 mmol) was dissolved in dry tetrahydrofuran, triphenylphosphoranylideneacetic acid methyl ester (12 mmol) was added under an argon stream, and the mixture was stirred at room temperature for 18 hours. Saturated brine and ethyl acetate were added to the reaction solution, and the ethyl acetate layer was washed twice with saturated brine and then concentrated to dryness. This residue was subjected to catalytic hydrogenation in the presence of ethyl acetate and 5% palladium / carbon in a hydrogen gas stream. After completion of hydrogenation, palladium / carbon was filtered off and concentrated to dryness. The residue was hydrolyzed with dilute hydrochloric acid to obtain 3- (2,5-dihydroxy-phenyl) -propionic acid.
On the other hand, after dispersing 1.0 g of platinum particles whose surface was platinum oxide in acetonitrile, 3-aminopropyltrimethoxysilane (10 mmol) was added, and the mixture was stirred at room temperature for 24 hours under an argon stream. The platinum particles were filtered off, washed 5 times with acetonitrile, and then again immersed in acetonitrile. Next, succinic anhydride (10 mmol) and triethylamine (10 mmol) were added and stirring was continued for 4 hours at room temperature. After completion of the reaction, the platinum particles were filtered off and further washed 5 times with acetonitrile. Subsequently, the obtained platinum particles were further dispersed in fresh acetonitrile, thionyl chloride (10 mmol) was added, and the mixture was stirred at room temperature for 3 hours under an argon stream. After completion of the reaction, the platinum particles were filtered and washed, and immediately, the platinum particles were immersed in an acetonitrile solution containing 3- (2,5-dihydroxy-phenyl) -propionic acid (10 mmol), stirred, and left for 16 hours. Finally, the platinum particles were filtered and washed. Next, after immersing the platinum particles in acetonitrile, 1,6-diaminohexane (10 mmol) and dicyclohexylcarbodiimide (20 mmol) were added, and the mixture was stirred at room temperature for 10 hours. After completion of the reaction, the platinum particles were filtered and washed to obtain a linker having an electrolytic cleavage unit bonded to the target gold-platinum oxide particles via silicon atoms. The platinum particles thus obtained have an amino group at the terminal, and any ligand can be bound to the amine.
Example 4 Benzoylation was performed by reacting an electrochemically cleaved terminal amine in aqueous solution with benzoyl chloride as a model of the ligand. After the chemically modified gold particles were dispersed in dimethylformamide, an excess amount of benzoyl chloride was added. Stir at room temperature for 3 hours. After cooling the reaction vessel with ice, methanol was added dropwise. Thereafter, the platinum particles were separated, washed 5 times with acetonitrile, and further washed 3 times with pure water. The obtained platinum particles were packed in a column tube as shown in FIG. 2, and after setting a platinum conductor, 50 mM phosphate buffer (pH 7.2) was filled. Next, with the platinum particle side as the anode, the potential was repeatedly swept between the terminal voltage of 0 V and 1.6 V (reciprocating sweep, 100 mV / sec). After 40 sweeps, the lead was disconnected and the buffer was eluted from the column. From this buffer solution, the ligand unit cleaved at the phenyl ester moiety by electrolysis was recovered.
Embodiment 5 FIG. The following formula was prepared by binding to a purified gold surface by affinity adsorption-electrolytic cleavage of biological material :
2.0 g of gold particles having a linker of 2 were dispersed in 10 ml of dry dimethylformamide. By adding 20 mg of biotin and 100 mg of dicyclohexylcarbodiimide to this dispersion, and stirring for 24 hours at room temperature under an argon stream, the following formula:
The ligand conjugate shown in FIG. This dispersion was filtered, washed 5 times with acetonitrile, and then dispersed in a phosphate buffer. The gold particles bound with biotin thus obtained were packed in a column. At this time, a conducting wire and a counter electrode were set. Next, 10 ml of a phosphate buffer (pH 7.2) containing 2 mg of the glycoprotein avidin to be separated was passed (flow rate: about 0.2 ml / min). Subsequently, a washing operation was performed at the same flow rate using 40 ml of a phosphate buffer containing no biomolecule, and it was confirmed by ultraviolet absorption spectrum and high performance liquid chromatography that the protein was not eluted in the washing solution. Next, the avidin-biotin complex was eluted by electrolysis. At this time, the same phosphate buffer was used as the eluent, and the potential sweep was repeated in the range of 0 to 1.5 volts while supplying the eluate to the column. At this time, the eluate was collected, and elution of the complex was confirmed by ultraviolet absorption and high performance liquid chromatography.
Example 6 Production of Platinum Particle Affinity Carrier An electrocleavable linker was synthesized from paramethoxybenzaldehyde and 3-mercaptopropyltrimethoxysilane according to the scheme shown in FIG. This linker was dissolved in n-butanol, 1 g of platinum particles having a particle size of 1-10 μm whose surface was platinum oxide was added, and the linker was bonded to the surface of the platinum oxide by breaking the trialkoxysilyl group. Each silyl group was bonded to the platinum surface via 1 to 3 oxygen atoms. By energizing the platinum carrier as an anode in the electrolytic solution, the benzyldithioacetal site was selectively cleaved oxidatively, and the cleaved aldehyde was eluted.
Example 7 Electrolytic cleavage using platinum particle affinity carrier The electrocleavable linker produced in Example 6 is reacted with 4-bromobutanoic acid to introduce a carboxyl group at the terminal, and a dye (Azure A) is bound as a ligand model to this. It was. The experiment was performed using the apparatus shown in FIG. The column was filled with platinum oxide particles bound with a dye, filled with an electrolytic solution (1M aqueous solution of lithium perchlorate) and allowed to stand for 10 minutes, and then the electrolytic solution was removed by suction. The column was washed three times with an electrolytic solution (1M aqueous lithium perchlorate). The washing solution was transparent and it was confirmed that the dye component did not elute. Next, the electrolytic solution was filled and energized (1.5 V vs Ag / AgCl, 5 minutes) using the platinum carrier as an anode. The blue pigment was eluted by suction filtration. That is, the benzyldithioacetal site was selectively cleaved oxidatively, and the cleaved aldehyde (blue dye) was eluted. When the electrolysis time was 30 seconds, the amount of the dye per gram of platinum was 0.1 × 10 −5 mol as judged by the absorbance of the eluted dye. The dye eluted after 5 minutes of energization was 0.8 × 10 −5 mol per gram of platinum. This clearly showed that the pigment component was cut and eluted by energization, that is, electrolytic oxidation.
Example 8 FIG. Production of platinum particle affinity carrier having biotin as a ligand The biotin-bound platinum particle affinity carrier shown in FIG. 12 was synthesized. The synthesis method followed the scheme described in FIG. The obtained biotin-coupled electrolytic cleavage affinity carrier was packed into a column, and an avidin aqueous solution was allowed to flow through the column, followed by washing with pure water and an electrolyte solution. Next, 1.5 V vs Ag / AgCl, a potential was applied to the affinity carrier for 5 minutes, and then the electrolyte in the column was eluted. The eluate was analyzed by electrophoresis to confirm that avidin was eluted (FIG. 14).

Claims (11)

  1. A method for purifying biological material comprising:
    A method comprising recovering the biological material by contacting an aqueous solution containing the biological material with an electrocleavable affinity carrier, and applying a voltage to the aqueous solution to cleave the electrolytically cleavable affinity carrier.
  2. The method of claim 1, wherein the electrocleavable affinity carrier is composed of a ligand bound to a solid support via an electrocleavable linker.
  3. The method of claim 2, wherein the electrocleavable linker is bound to a solid support by a platinum oxide-silicon bond.
  4. The electrolytic cleavable linker is
    Electrolytic cutting part,
    The method of claim 2, comprising at least one ligand binding arm coupled to the electrolytic cleavage moiety and at least one carrier binding arm coupled to the electrolytic cleavage moiety.
  5. An electrocleavable affinity carrier which is an electrocleavable affinity carrier which is cleaved by applying a voltage, the electrocleavable affinity carrier comprising a ligand bound to a solid carrier via an electrocleavable linker.
  6. The electrolytically cleavable affinity carrier according to claim 5, wherein the electrolytically cleavable linker is bound to a solid carrier by a platinum oxide-silicon bond.
  7. The electrolytic cleavable linker is
    Electrolytic cutting part,
    6. The electrocleavable affinity carrier according to claim 5, comprising at least one ligand binding arm bound to the electrolytic cleavage moiety and at least one carrier binding arm bound to the electrolytic cleavage moiety.
  8. A biological material purification kit comprising the electrocleavable affinity carrier according to claim 5, 6 or 7.
  9. An electrocleavable linker that is cleaved by applying a voltage,
    Electrolytic cutting part,
    Electrolytic cleavable linker comprising at least one ligand binding arm bound to the electrolytic cleavage moiety and at least one carrier binding arm bound to the electrolytic cleavage moiety.
  10. 10. The electrocleavable linker of claim 9, further comprising at least one carrier bound via the carrier binding arm.
  11. The electrocleavable linker according to claim 10, wherein the carrier binding arm is bonded to a solid support by a platinum oxide-silicon bond.
JP2004530574A 2002-08-21 2003-08-20 Biomaterial purification method using electrocleavable affinity carrier, electrocleavable affinity carrier, biomaterial purification kit, electrocleavable linker Expired - Fee Related JP4135791B2 (en)

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