JP2005312425A - Method for immobilizing polypeptide and solid support having polypeptide immobilized thereon, method for detection and purification of polypeptide using the same and solid support for immobilizing polypeptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for strongly and specifically immobilizing a polypeptide and to provide a solid support for the same. <P>SOLUTION: This invention relates to the method for immobilizing the polypeptide comprising bonding the polypeptide having an oligocysteine sequence introduced thereinto, to the solid support having a group represented by formula I on the surface through the reaction with its maleimido groups, wherein R is a 1-12C divalent hydrocarbon group. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for immobilizing a polypeptide, a solid support on which the polypeptide is immobilized, a method for detecting and purifying a polypeptide using the same, and a solid support for immobilizing the polypeptide. .

  With the completion of the Human Genome Project, biological and medical research has taken a new step from genetic decoding to protein analysis, or proteomics. A gene is simply a program code for producing a protein, and almost all biological activities are performed between molecules of a protein having a very complicated structure generated based on the code. It is known that if certain proteins do not function properly, they can interfere with health. Therefore, elucidating the functions of individual proteins can be said to be an indispensable step for further progress in medicine.

  Conventionally, two-dimensional electrophoresis (2-D PAGE) that separates and analyzes a mixture of extracted proteins based on differences in molecular weight or isoelectric point is used for analysis of protein properties, expression state, structure, activity, and the like. Have been used. However, the two-dimensional electrophoresis method is not suitable for high-throughput analysis and has problems in terms of detection sensitivity and sample solubilization.

  On the other hand, many DNA chips have been reported to date. These DNA chips were useful for confirming changes in gene expression in a certain phenotype or physiological state and for creating a database of expression patterns. However, since the expression levels and patterns of genes and proteins do not always accurately correlate, DNA chips cannot be used for quantification of protein expression levels. In addition, since the function of a protein is changed by undergoing various modifications such as phosphorylation, sugar chain addition, and cleavage after translation, information on the post-translational modification cannot be obtained from DNA analysis.

  Therefore, a protein chip has been developed using DNA array technology as a tool for protein analysis. The principle of a protein chip is the same as that of a DNA chip. Proteins are immobilized on a slide glass or membrane at a high density, and proteins or nucleic acids that interact with them are detected. However, it has been difficult to immobilize on a substrate a protein having a very unstable structure, which is formed by tangling amino acids with a strong DNA chain.

  In addition, conventionally developed protein chips are those in which a polymer such as polylysine is applied to the surface of a slide glass or silicon substrate, and then proteins are immobilized. In the method of immobilizing proteins by applying the protein, the protein immobilization state is unstable, causing problems such as peeling during the washing process, and it is impossible to store the immobilized proteins for a long period of time. It was. In addition, there are problems such as non-specific adsorption of biological substances, protein function inactivation, and detection system UV opacity.

  In view of such problems, as a support for immobilizing a protein-containing polypeptide, a solid support (for example, Patent Document 1) in which a surface treatment layer and a chemical modification layer are sequentially provided on the surface of a substrate, A solid support (Patent Document 2) in which a part or all of the substrate surface is composed of a carbon layer and a metal chelate is formed on the layer has been developed.

  However, the amount of polypeptide immobilized on the solid support and the binding strength of the polypeptide are not necessarily sufficient, and the appearance of a solid support having a higher amount of immobilized polypeptide and higher polypeptide binding strength has emerged. It is desired.

Prior art document information relating to the invention of this application includes the following.
JP 2002-365293 A JP 2004-020328 A

  An object of the present invention is to provide a method for firmly and specifically immobilizing a polypeptide, and a solid support therefor.

As a result of intensive studies to solve the above problems, the present inventors have used a solid support on which a maleimide group has been introduced via an amino group on a substrate and introduced an oligocysteine sequence into the polypeptide, As a result, it was found that the immobilization amount and the binding strength of the polypeptide were remarkably improved, and the present invention was completed.
That is, the present invention includes the following inventions.

［式中、Ｒは鎖員１〜１２の２価の炭化水素基である］
で表される基を有する固体支持体のマレイミド基に、オリゴシステイン配列が導入されたポリペプチドを結合させることにより、ポリペプチドを固定化する方法。 (1) The formula I:

[Wherein R is a divalent hydrocarbon group having 1 to 12 chain members]
A method for immobilizing a polypeptide by binding a polypeptide having an oligocysteine sequence introduced thereto to a maleimide group of a solid support having a group represented by the formula:

(2) The method according to (1), wherein the substrate further has a carbon layer.

［式中、Ｒは鎖員１〜１２の２価の炭化水素基である］
で表される基を有する固体支持体のマレイミド基に、オリゴシステイン配列が導入されたポリペプチドが結合されている、ポリペプチド固定化固体支持体。 (3) Formula I on the surface of the substrate:

[Wherein R is a divalent hydrocarbon group having 1 to 12 chain members]
A polypeptide-immobilized solid support, wherein a polypeptide into which an oligocysteine sequence is introduced is bound to a maleimide group of the solid support having a group represented by

(4) The polypeptide-immobilized solid support according to (3), wherein the substrate further has a carbon layer.
(5) The polypeptide-immobilized solid support according to (3) or (4), wherein an oligohistidine sequence is further introduced into the polypeptide into which the oligocysteine sequence has been introduced.
(6) A method for detecting the interaction between the immobilized polypeptide and the sample polypeptide by bringing the sample polypeptide into contact with the polypeptide-immobilized solid support according to any one of (3) to (5).
(7) The method according to (6), wherein the interaction is detected by labeling the sample polypeptide and detecting a label derived from the sample polypeptide that has interacted with the immobilized polypeptide.
(8) The method according to (7), wherein the interaction of the polypeptide is detected using chemiluminescence.

［式中、Ｒは鎖員１〜１２の２価の炭化水素基である］
で表される基を有する固体支持体上のマレイミド基と反応させ、オリゴシステイン配列が導入されたポリペプチドを固体支持体上に結合させることによって、該ポリペプチドを精製する方法。 (9) An oligocysteine sequence is introduced into the polypeptide, and the polypeptide into which the oligocysteine sequence is introduced is represented on the surface of the substrate by formula I:

[Wherein R is a divalent hydrocarbon group having 1 to 12 chain members]
A method for purifying a polypeptide by reacting with a maleimide group on a solid support having a group represented by the following formula, and binding the polypeptide introduced with an oligocysteine sequence onto the solid support.

(10) The method according to (9), wherein the substrate further has a carbon layer.

［式中、Ｒは鎖員１〜１２の２価の炭化水素基である］
で表される基を有する、ポリペプチドを固定化するための固体支持体。 (11) Formula I on the surface of the substrate:

[Wherein R is a divalent hydrocarbon group having 1 to 12 chain members]
A solid support for immobilizing a polypeptide having a group represented by the formula:

(12) The solid support according to (11), wherein the substrate further has a carbon layer.
(13) A vector for producing a gene encoding a polypeptide into which an oligocysteine sequence and an oligohistidine sequence are introduced.
(14) A polypeptide into which an oligocysteine sequence and an oligohistidine sequence are introduced.

  According to the present invention, a polypeptide can be firmly and specifically immobilized on a solid support. Therefore, a protein chip with a high detection limit can be produced by using the solid support of the present invention. Using such a protein chip, a polypeptide that interacts with the immobilized polypeptide can be detected with high sensitivity.

Solid support In the present invention, the solid support on which the polypeptide is immobilized has a structure in which a chemically modifying group is bonded to the surface of the substrate.

  Examples of the material of the substrate used in the present invention include silicon, glass, fiber, wood, paper, ceramics, plastic (for example, polyester resin, polyethylene resin, polypropylene resin, ABS resin (Acrylonitrile Butadiene Styrene resin), nylon, and acrylic resin. , Fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin). In the present invention, it is preferable to use a silicon substrate.

  In the present invention, it is desirable to form a carbon layer on this substrate. The carbon layer formed on the substrate in the present invention is not particularly limited, but diamond, diamond-like carbon, amorphous carbon, graphite, hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide. , Tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, vanadium carbide and the like, and a diamond-like carbon (DLC) layer is preferable.

  The carbon layer has excellent chemical stability and can withstand subsequent chemical modification and reaction in the binding to the analyte, and the binding is flexible because it binds to the analyte by electrostatic binding. and it has a point, that it is transparent to the detection system UV because no UV absorption, and is advantageous in terms that can be energized during electroblotting. Further, in the coupling reaction with the analyte, is advantageous in that non-specific adsorption is small.

  In the present invention, the carbon layer can be formed by a known method. For example, a microwave plasma CVD (Chemical vapor deposition) method, an ECRCVD (Electrical cyclotron resonance) method, an ICP (Inductive coupled plasma) method, a direct current sputtering method, an ICP (Inductive coupled plasma) method, a direct current sputtering method, a direct current sputtering method. Examples thereof include a vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method.

  In the high-frequency plasma CVD method, a source gas (methane) is decomposed by glow discharge generated between electrodes by a high frequency to synthesize a DLC (diamond-like carbon) layer on a substrate. In the ionization vapor deposition method, the source gas (benzene) is decomposed and ionized using thermoelectrons generated by a tungsten filament, and a carbon layer is formed on the substrate by a bias voltage. The DLC layer may be formed by ionized vapor deposition in a mixed gas composed of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of remaining methane gas.

  In the arc evaporation method, an arc discharge is generated in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode) to generate a plasma of carbon atoms from the cathode, thereby generating an evaporation source. Further, by applying a negative bias voltage to the substrate, carbon ions in the plasma can be accelerated toward the substrate to form a carbon layer.

  In the laser vapor deposition method, for example, a carbon layer can be formed by irradiating a graphite target plate with Nd: YAG laser (pulse oscillation) light and melting it, and depositing carbon atoms on a glass substrate.

  In the present invention, the thickness of the carbon layer is usually a monomolecular layer to about 100 μm. If it is too thin, the surface of the underlying solid support may be locally exposed. Therefore, it is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm. Note that all of the solid support may be made of a carbon material.

  In the present invention, the solid support not only has a structure in which a carbon layer is formed on a substrate as described above, but also a laminate or composite of diamond-like carbon and a substrate material (for example, diamond-like carbon and other substances). Complex (for example, two-phase body)).

  By forming a carbon layer such as a diamond-like carbon layer on the substrate, the polypeptide can be immobilized at a high density and a high S / N ratio can be obtained, so that highly sensitive detection is possible. It can also be used repeatedly.

  Although the shape and size of the substrate are not particularly limited, examples of the shape include a flat plate shape, a thread shape, a spherical shape, a polygonal shape, a powder shape, and the size is usually 0.1 mm in width when a flat plate shape is used. ˜100 mm, length 0.1˜100 mm, thickness 0.01˜10 mm.

  When glass is used as the substrate, the surface thereof is preferably intentionally roughened in the range of 1 nm to 1000 nm with Ra (JIS B 0601). Such a roughened surface is advantageous in that the surface area of the substrate is increased and a large amount of polypeptide can be immobilized at a high density.

  Subsequently, a solid support is produced by introducing a chemical modifying group into the substrate having the carbon layer. Introduction of the chemical modification group can be carried out as follows.

  First, an amino group is introduced into the substrate having the carbon layer. The amino group can be introduced by chlorinating the surface by irradiating the substrate with ultraviolet rays in chlorine gas and then irradiating the substrate with ultraviolet rays in ammonia gas. Or it can implement by giving the board | substrate which gave the diamond-like carbon layer to a plasma method in ammonia atmosphere. Here, the plasma method is a method in which plasma is generated in a discharge by direct current or alternating current under vacuum conditions, for example, benzene or methane is used as a source gas, and a substrate to which a bias is applied with an ionized gas is processed.

で表される化合物又はその塩を反応させることにより、式I：

で表されるような化学修飾基をカーボン層上に形成することができる。 The amino group thus formed and the following formula II:

Is reacted with a compound represented by the formula:

Chemically modified groups as represented by can be formed on the carbon layer.

In Formula I or II, R is a divalent hydrocarbon group having 1 to 12 chain members. The divalent hydrocarbon group having 1 to 12 chain members is an alkylene group having 1 to 12, preferably 4 to 6 chain members, such as a tetramethylene group, a pentamethylene group, a hexamethylene group, or a chain member 1 to 12. And a divalent alicyclic hydrocarbon group such as a cyclohexylene group, an arylene group such as a 2,2′-biphenylene group, and a divalent aromatic ring-containing hydrocarbon group such as an o-xylylene group. These divalent hydrocarbon groups may be substituted with, for example, a C _1-10 -alkyl group, an aryl group, a trimethylsilyl group, an acyl group, and the like.

Here, as the C _1-10 -alkyl group, for example, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, hexyl group , Heptyl group, octyl group, nonyl group, decyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, and cyclohexyl group. Examples of the aryl group include aromatic hydrocarbon groups such as phenyl group, 1-naphthyl group, and 2-naphthyl group, furyl group, thienyl group, pyrrolyl group, oxazolyl group, isoxazolyl group, thiazolyl group, isothiazolyl group, and imidazolyl group. And aromatic heterocyclic groups such as a pyrazolyl group, a pyridyl group, a pyrimidinyl group, a pyridazinyl group, a pyrazinyl group, a quinolyl group, and an isoquinolyl group. Examples of the acyl group include C _1-10 -aliphatic acyl groups such as formyl group, acetyl group, propanoyl group, butanoyl group, pentanoyl group and hexanoyl group.

Preferable specific examples of R include — (CH ₂ ) ₄ —, — (CH ₂ ) ₅ —, — (CH ₂ ) ₆ —, —CH (CH ₃ ) — (CH ₂ ) ₃ —, —CH ₂ CH _{(CH 3) - (CH 2} ) 2 -, - CH (CH 2 CH 3) - (CH 2) 3 -, - CH (CH 2 CH 2 CH 3) - (CH 2) 3 -, - CH (CH _{3) - (CH 2) 4} -, - CH (CH 2 CH 3) - (CH 2) 4 -, - CH (CH 2 CH 2 CH 3) - (CH 2) 4 -, - CH 2 CH (CH _{2 CH 2 CH 3) - (} CH 2) 3 -, - CH (CH 3) - (CH 2) 5 -, - CH (CH 2 CH 3) - (CH 2) 5 -, - CH (CH 2 CH _{2 CH 3) - (CH 2} ) 5 -, - C 6 H 4 -, - C 6 H 4 -CH 2 -, - C 6 H 4 - (CH ₂ ) _2— , —C ₆ H ₃ (CH ₃ ) — and the like.

  Although it does not specifically limit as a salt of the compound represented by Formula II, For example, a sodium salt, potassium salt, etc. can be used. The sodium salt is preferably used.

  Specifically, the reaction is performed by immersing the solid support into which the amino group has been introduced in a solution containing the compound of formula II in a buffer usually at a concentration of 0.1 to 100 mM. As the buffer, PBS (phosphate buffered saline), triethanolamine buffer, sodium borate buffer, or the like can be used. It is preferable to use PBS (pH 6-9). The reaction temperature is usually 10 to 80 ° C., preferably 25 to 30 ° C., and the reaction time is usually 1 to 300 minutes, preferably 30 to 60 minutes.

  In the solid support of the present invention, the above-mentioned chemically modifying group having a maleimide group is firmly bonded to the carbon in the carbon layer by a covalent bond, so that it does not peel off due to washing or temperature change. It is also possible to store the support for a long time.

Immobilization of polypeptides In the present invention, polypeptides include peptide fragments, oligopeptides and proteins. It may be a simple protein or a complex protein, and may be natural or synthetic. The number of amino acids of the polypeptide immobilized on the solid support of the present invention is not particularly limited, but is usually 10 to 3000, preferably 50 to 2000, and more preferably 100 to 1500. The polypeptide that can be immobilized on the solid support of the present invention is not particularly limited, and examples thereof include antibodies, enzymes, pathogenic proteins, peptide hormones, receptors, kinases, glycoproteins, metalloproteins, viral proteins, derived proteins, and the like. Can be suitably immobilized.

  In the present invention, the polypeptide includes peptide derivatives. Peptide derivatives include peptides in which one or several amino acids are derivatized by a chemical reaction. Examples of peptide derivatives include molecules in which a reactive amino acid side group such as a free amino group, a free carboxyl group or a free hydroxyl group is derivatized. Specific examples of derivatives of amino groups are sulfonic acid or carboxylic acid amides, thiourethane derivatives and ammonium salts such as hydrochloride. Examples of carboxyl group derivatives are salts, esters and amides. Examples of hydroxyl group derivatives are O-acyl or O-alkyl derivatives. Also included are peptides in which one or several amino acids are replaced by an amino acid homologue of 20 “standard” amino acids, which are naturally occurring or non-naturally occurring. Examples of such homologues are 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine, ornithine, β-alanine and 4-aminobutyric acid.

  In the present invention, an oligocysteine sequence is introduced into the polypeptide, and a covalent bond is formed by reacting the maleimide group present on the surface of the solid support of the present invention with the SH group of the cysteine residue. Since the maleimide group has high selectivity for the SH group, a polypeptide having an oligocysteine sequence introduced can be specifically bound on a solid support.

  In the present invention, the oligocysteine sequence usually means a sequence in which at least 3, preferably 3 to 10, more preferably 4 to 6 cysteine residues are continuous. In the present specification, the oligocysteine sequence may be referred to as a cysteine tag.

  In the present invention, it is preferable to introduce a label for further purification into the target polypeptide in addition to the oligocysteine sequence for immobilization. Such labels include oligohistidine sequences that specifically interact with metal ions such as zinc, nickel or cobalt ions (referred to as histidine tags, such as hexahistidine sequences), or zinc or Examples include polylysine sequences containing at least about 4 lysines or polyarginine sequences containing at least 4 arginine residues that interact specifically with copper. By introducing such a label, the polypeptide of interest can be easily isolated from the translation reaction product using a reagent or column that specifically interacts with the label. For example, a nickel column can be used for the oligohistidine sequence.

  Introduction of the oligocysteine sequence and oligohistidine sequence into the polypeptide can be performed by a method combining PCR generally used in the art and gene cloning using Escherichia coli. For example, a plasmid vector having a region encoding an oligocysteine sequence and an oligohistidine sequence is constructed in advance, a target polypeptide is introduced later using a restriction enzyme site, etc., and a host cell is transformed with the obtained recombinant vector. By converting and expressing the fusion polypeptide in the transformant, a polypeptide in which oligocysteine and oligohistidine sequences are linked can be produced.

  Alternatively, when a nucleotide sequence encoding a target polypeptide is amplified by PCR or the like, a nucleotide sequence encoding an oligocysteine sequence and an oligohistidine sequence is contained in each DNA primer, and this is used to target the target polypeptide. This DNA fragment can be introduced into a vector after being amplified and expressed to produce a fusion polypeptide linked to both sequences.

  The oligocysteine sequence is generally introduced at the N-terminal or C-terminal of the target polypeptide, but the introduction site is not limited to the terminal as long as it does not hinder immobilization. Since the SH group reacts with the maleimide group to form a covalent bond, the target polypeptide binds tightly to the chemically modified group of the present invention.

  The oligohistidine sequence is generally introduced in a form linked to the N-terminus or C-terminus of the target polypeptide that does not have the oligocysteine sequence, or linked to the oligocysteine sequence. The introduction site is not limited to the end.

  A target polypeptide having oligohistidine and oligocysteine sequences is expressed and extracted in a host cell, and then purified by a conventional method such as using a nickel column using an oligohistidine sequence.

  The polypeptide into which the oligocysteine sequence has been introduced can be dissolved in a spotting buffer and spotted on the solid support of the present invention to immobilize the polypeptide.

  A polypeptide into which an oligocysteine sequence has been introduced is dissolved in a spotting buffer so that the concentration is usually 0.01-100 μM, preferably 5-50 μM, to prepare a spotting solution. The spotting buffer is 1 to 100%, preferably 20 to 50% PEG (polyethylene glycol) solution, PBS (phosphate buffered saline), 50% DMSO (dimethyl sulfoxide), 3 × SSC (saline sodium). citrate), pure water or the like can be used. In the present invention, it is preferable to use a 50% PEG solution. Maleimide groups are easily hydrolyzed and have the best reaction efficiency around pH 7.

  The prepared spotting solution can be dispensed into a 96-well or 384-well plastic plate, and the dispensed solution can be spotted on a solid support by a spotter device or the like. At this time, by arranging multiple types of polypeptides in an array as independent spots, the interaction of the multiple types of polypeptides can be detected.

  After spotting, incubation is preferably performed in order to allow the reaction of the polypeptide to bind to the solid support. Since the immobilization of the polypeptide is the production of a covalent bond by a chemical reaction, it is preferable to perform incubation immediately after spotting in order to ensure the reaction proceeds.

  Incubation is usually performed at a temperature of 4 to 30 ° C., preferably 25 to 30 ° C., usually for 0.5 to 16 hours, preferably for 1 to 2 hours. Incubation is preferably performed under a high humidity atmosphere, for example, under conditions of a humidity of 50 to 90%. Following the incubation, washing is preferably performed using a washing solution (eg 50 mM TBS / 0.05% Tween 20) to remove the polypeptide that is not bound to the solid support.

Detection of Peptide Interaction The present invention also relates to a method for detecting an interaction between polypeptides by reacting a polypeptide immobilized on a solid support as described above with a sample polypeptide that interacts with the polypeptide. . Examples of the interaction between polypeptides include antigen-antibody reaction, enzyme reaction, receptor-ligand reaction, biotin-streptavidin interaction, and the like.

  The presence or absence of interaction can be detected by labeling the sample polypeptide, bringing it into contact with the polypeptide immobilized on the solid support, and reading the label derived from the sample polypeptide that has interacted with the polypeptide. . The labeling of the sample polypeptide can be performed by a method usually used in the art.

  The label is not particularly limited as long as it can be incorporated into the polypeptide. For example, fluorescent labels (CyDye such as Cy3 and Cy5, FITC, RITC, rhodamine, Texas red, TET, TAMRA, FAM, HEX, ROX, GFP, etc.), radioactivity labels (α-32P, γ-32P, 35S, etc.), enzyme labels (HRP (Horserishing peroxidase), alkaline phosphatase, etc.). When a fluorescently labeled polypeptide is used, it can be detected as an image by fluorescence imaging of the solid support after the interaction. When an enzyme label is used, a chemiluminescent reagent (luminol, luciferin, umbelliferone, coelenterazine, dioxycetane compound, D-luciferin potassium, bis (2,4,6-) oxalate is added to the solid support after the interaction. It can be detected by the action of trichlorophenyl)).

  The sample polypeptide sample is prepared by dissolving in a buffer so that the concentration is usually 0.01-100 μM, preferably 1-10 μM. The obtained sample solution is dropped on a solid support on which the polypeptide prepared above is immobilized, and allowed to interact by incubation.

  Incubation is usually carried out at 4 to 30 ° C., preferably 25 to 30 ° C., usually for 0.5 to 16 hours, preferably for 1 to 4 hours. Also in the step of interacting, it is preferable to carry out incubation in a high humidity atmosphere. Finally, the solid support is washed, dried, and then detected by reading the label.

  Alternatively, by performing mass spectrometry of the complex formed by the interaction, the amino acid sequence of the polypeptide that specifically interacts with the polypeptide on the solid support can be analyzed. Alternatively, mass spectrometry can be performed by ionizing only the polypeptide that interacts with the polypeptide immobilized on the solid support.

  Immobilized substance on a solid support, laser desorption / ionization - can be carried out as mass analyzed by means such as time-of-flight mass spectrometry. Mass The mode of ionization that can be used to analyze, matrix-assisted laser desorption (MALDI) method, ionization by electron impact (EI) method, a photo ionization method, large α or β rays LET emitted from radioisotopes ionization method using secondary ionization, fast atom bombardment ionization method, field ionization ionization, surface ionization ionization, chemical ionization (CI) method, field ionization (FI) technique, ionization method, and the like due to the spark discharge , matrix-assisted laser desorption (MALDI) method, ionization by electron impact (EI) method is preferred.

  The present invention also introduces an oligocysteine sequence into the target polypeptide, contacts it with the solid support of the present invention, and binds it to the solid support by covalent bond with a maleimide group. The present invention relates to a method for purification Since the solid support having a maleimide group of the present invention specifically binds to an SH group, it can be separated and purified by binding only a polypeptide into which an oligocysteine sequence has been introduced.

In the present invention, a solid support in which a protein expression vector for expression in a form in which an oligocysteine sequence is fused to a protein is constructed, and a maleimide group specifically covalently bonded under mild conditions is introduced into the SH group of cysteine Can be used to recover the protein. Using a protein tag consisting of an oligocysteine sequence and using a covalent bond as a recovery system includes a histidine tag that uses a conventional metal chelate, a GST tag that uses the affinity between the enzyme and the substrate, and a FLAG that uses a specific antibody. It is based on a new concept that is completely different from epitope tags. Whereas the conventional protein tag was focused on purifying the tagged protein itself, the cysteine tag (oligocysteine sequence) in the present invention is recovered by a strong covalent bond. It is possible to widen the setting range of separation and recovery conditions for the binding protein separated together with the introduced protein. Therefore, it can be expected that the binding protein group that could not be collected so far will be picked up and the protein interaction analysis will be greatly improved. In addition, the covalent recovery facilitates washing and purification of the cysteine-tagged protein itself, and is an excellent system for analyzing post-translational modifications of proteins by subjecting them to mass spectrometry after protease cleavage on a solid support. Can provide.
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these.

Example 1. Preparation of solid support with maleimide group introduced DLC layer at an acceleration voltage of 0.5 kV using a mixture of 95% by volume of methane gas and 5% by volume of hydrogen on a silicon substrate cut into 3 mm square by ionization vapor deposition Was formed to a thickness of 100 nm. Thereafter, amination was performed for 10 minutes by a plasma method in an ammonia gas atmosphere.

A reaction solution having the following composition was prepared, and the aminated solid support was immersed therein and infiltrated for 30 minutes. Then, it wash | cleaned 3 times with pure water, and vacuum-dried at 100 degreeC for 30 minutes. Here, sulfo-EMCS (Dojindo Laboratories Co., Ltd.) is a sodium salt in which R is — (CH ₂ ) ₅ — in the compound of the above formula II.

Sulfo-EMCS 12.3 mg [1 mM]
10 x PBS (pH 7.4) 3ml
27ml of ultrapure water
30ml total

  The surface of the solid support before and after the reaction was analyzed using micro-ESCA (Electron spectroscopy for chemical analysis: manufactured by Perkin-Elmer). The results are shown in FIG.

  From the ESCA analysis of the solid support surface before and after the reaction, it was found that the peak intensities of O and N increased after the reaction (FIG. 1a). Further, it can be seen from the narrow scan after the reaction that a new peak appears in the vicinity of about 289 eV (FIG. 1b). The O and N peaks are considered to be derived from O and N of the maleimide group. Moreover, it is considered that the peak newly appearing in the vicinity of 289 eV is derived from the imide portion of maleimide. From the above, it can be seen that maleimide groups are introduced on the surface of the solid support.

Example 2 Expression and purification of EGFP (Enhanced Green Fluorescence protein) introduced with oligocysteine sequence <expressed protein>
Sample 1: 6 × His (control without GFP gene)
Sample 2: 6 × His-EGFP (EGFP introduced with 6 histidines at the N-terminus)
Sample 3: 5 × Cys-EGFP (EGFP with 5 cysteine introduced at the N-terminus)
Sample 4: 5 × Cys-EGFP-6 × His (EGFP introduced with 5 cysteines at the N-terminus and 6 histidines at the C-terminus)
Sample 5: 6 × His-EGFP-5 × Cys (EGFP introduced with 6 histidines at the N-terminus and 5 cysteines at the C-terminus)
The structure of the protein represented in Samples 1 to 5 is shown in FIG.

<Construction of expression vector>
An outline of the construction of the expression vector is shown in FIG. First, based on pET14b (manufactured by Novagen), an expression plasmid vector for a His-tagged protein (a protein in which 6 histidines are introduced at the N-terminus), a PCR method and a gene cloning method in Escherichia coli are used to obtain a new downstream of the His tag. Cloning sites NdeI, BglII, XhoI, BamHI and KpnI were introduced (FIG. 3, Step 1). In the same manner, a 5 × Cys protein expression vector in which 6 histidines were replaced with 5 cysteines was constructed (FIG. 3, Step 2). The GFP gene of Clonetech pEGFP-C1 was cloned by PCR with the restriction enzyme sites NdeI and XhoI attached, inserted downstream of 6 × His and 5 × Cys with a reading frame, and 6 × His-GFP and A 5 × Cys-GFP (FIG. 3, Step 3) protein expression vector was constructed.

  In addition, two types of 5 × Cys- and 6 × His-double-tagged GFP (Cys-GFP-His and His-GFP-Cys) expression vectors were constructed. It was prepared by introducing 5 × Cys downstream of 6 × His-GFP and 6 × His downstream of 5 × Cys-GFP (FIG. 3, Step 4). The gene sequence of each tag part was confirmed by DNA sequencing. Each vector was transformed into Escherichia coli BL21 strain for protein expression, and a strain with good GFP expression was selected. As the base sequence encoding 5 × Cys, comparison was made using TGTTGTGTGTGTGTGT (SEQ ID NO: 1) and TGTTGCTGTTGCTTGT (SEQ ID NO: 2). Since both were similarly expressed well, only the plasmid having the former base sequence was fixed. It was subjected to a chemical test.

<Culture>
A single colony was picked up with a toothpick from an agar medium (LB, Ampicillin 50 μg / ml, Chloramphenicol 50 μg / ml) in which BL21 strain (manufactured by Novagen) transformed with an expression vector into which DNA encoding each protein was introduced was cultivated. In LB medium (Ampicillin 50 μg / ml). IPTG was added to a final concentration of 0.4 mM and cultured at 23 ° C. and 160 rpm for 24 hours.

<Extraction>
Each culture broth was collected by centrifugation (1000 g, 10 min, 4 ° C.). The LB medium was discarded, resuspended in PBS (pH 7.4), E. coli was washed with Vortex and collected again. Thereafter, the dry weight of E. coli was measured. Protein extraction reagent Bugbuster (Novagen) was added at 5 ml / g, Benzonase (Novagen) was added at 25 U / ml, and Lysozyme (Novagen) was added at 5 KU / ml and shaken at room temperature for 20 minutes. After centrifugation (1000 g, 10 min, 4 ° C.), the supernatant was collected and filtered through a 0.2 μm filter.

<Purification>
Since Samples 1, 2, 4, and 5 have a His tag, they were purified using a His-Trap column (Amersham). Thereafter, the mixture was desalted with Microcon YM-10 (Millipore) and concentrated. Since sample 3 cannot be purified, only desalting and concentration were performed.

<Confirmation>
Confirmation of EGFP expression was performed by SDS-PAGE (gel concentration: 12.5%) (FIG. 4). Moreover, the fluorescence was confirmed with a UV irradiation device (FIG. 5).

Example 3 Spotting Polypeptide on Solid Support Having Maleimide Group Various kinds of tagged EGFP recovered in Example 2 and 100% PEG were mixed at 1: 1 to prepare 10 μl of a spotting solution. Each spotting solution was spotted by 0.5 μl on the solid support produced in Example 1 with the arrangement shown in FIG. 6 using a micropipette. Placed in a wet box and incubated at room temperature for 1 hour. After washing with 50 mM TBS / 0.05% Tween 20 (25 ° C., 5 min), it was washed with PBS (pH 7.4) (25 ° C., 5 min). It was washed with pure water, covered with care so as not to dry, and measured with LAS-1000 (manufactured by Fuji Photo Film Co., Ltd.) (exposure 30 seconds). The results are shown in FIG.

Example 4 Immobilization by immersion of EGFP with various tags on a solid support having a maleimide group The EGFP with various tags recovered in Example 2 and 2 × PBS were mixed 1: 1 with a PCR tube to prepare 50 μl of an immersion solution. . The solid support produced in Example 1 was immersed in a tube and incubated at room temperature for 18 hours. After washing with PBS for 30 minutes, it was washed with pure water and covered with care not to dry, and measured with LAS-1000 (manufactured by Fuji Photo Film Co., Ltd.) (exposure 30 seconds). The results are shown in FIG.

  The results of Examples 3 and 4 showed that the solid support of the present invention selectively binds to the cysteine tag rather than the histidine tag.

Example 5 FIG. Detection of polypeptide interaction using chemiluminescence 5-1 Preparation of patterned solid support Immobilization of enhanced green fluorescence protein (EGFP) by immersion and diamond-like carbon (DLC) layer so that chemiluminescence can be easily confirmed In addition, an amino group and a maleimide group were formed in a pattern to prepare (FIG. 9).

  A resist was applied on a silicon substrate with a spin coater. Next, it was exposed and developed with a mask having a pattern as shown in FIG. Then, the DLC layer was formed and amino groups were introduced in the same manner as in Example 1. The obtained substrate was immersed in 1 mM sulfo-EMCS reagent in 1 × PBS (pH 7.4) for 1 hour at room temperature, washed with PBS (10 minutes) and ultrapure water (10 minutes × 2), and then dried. I let you.

  Subsequently, the resist was stripped using ethanol, and the ethanol was washed. After air blowing, it was vacuum dried at 100 ° C. for 1 hour.

5-2 Immobilization of EGFP and detection of interaction by chemiluminescence 6 × His-EGFP-5 × Cys prepared by the same method as in Example 2 (6 histidines at the N-terminus and 5 cysteines at the C-terminus) EGFP) or 6 × His-EGFP (EGFP with 6 histidines at the N-terminus) dissolved in a buffer (20 mM sodium phosphate (pH 7.4) /0.5 M NaCl / 0.5 M imidazole). , Protein solutions of 100 ng / μl, 10 ng / μl, 1 ng / μl, 0.1 ng / μl and 0.01 ng / μl were prepared. The solid support prepared in 5-1 was immersed in these solutions, and was allowed to stand at room temperature for 1 hour in the dark. Then, it was washed with 50 mM TBS / 0.05% Tween 20 for 5 minutes × 3 times. Further, it was washed with ultrapure water for 5 minutes, and fluorescence was measured with LAS-1000 (manufactured by Fuji Photo Film Co., Ltd.) (FIG. 10). Blocking was performed at room temperature for 1 hour using Blocking Reagent (manufactured by Roche). The obtained solid support was reacted with a primary antibody (monoclonal anti-green fluorescent protein (SIGMA)) diluted to 1/10000 (room temperature, 1 hour). Washed with PBS for 10 minutes followed by 20 minutes. And it was made to react with the HRP labeled secondary antibody (HRP labeled anti-mouse IgG antibody (SIGMA)) diluted to 1/10000 (room temperature, 1 hour). Washed with PBS for 10 minutes → 20 minutes → 10 minutes. Chemiluminescence was measured using Super Signal ELISA Femto Maximum Sensitivity Substrate (Pierce) as a chemiluminescent reagent, and chemiluminescence was measured with LAS-1000 (Fuji Photo Film) (FIG. 11).

  From the above results, it was revealed that the polypeptide can be selectively bound to the solid support by the presence of the oligocysteine sequence. In addition, since GFP is bound only to the pattern forming portion into which the chemically modifying group is introduced, it has been clarified that nonspecific adsorption can be effectively prevented by the present invention.

  Furthermore, the polypeptide can be immobilized on the solid support by immersing the solid support in a low-concentration polypeptide solution of 3.7 fmol / μl, and the interaction of the polypeptide can be detected with high sensitivity by chemiluminescence. It became clear that we could do it.

The result of having carried out the ESCA analysis of the surface of the solid support body of the present invention created in Example 1 is shown. The structures of the five types of proteins expressed in Example 2 are shown. The outline | summary of the expression vector construction in Example 2 is shown. The result of having confirmed the expression of EGFP by SDS-PAGE is shown. The result of having confirmed the fluorescence with the UV irradiation apparatus is shown. The spotting positions of various polypeptides in Example 3 are shown. In Example 3, the result of having immobilized various polypeptides on a solid support having a maleimide group by spotting is shown. In Example 4, the result of immersing and immobilizing a solid support having a maleimide group in a solution containing various polypeptides is shown. In Example 5, it is a figure showing the pattern of the part which formed the DLC layer and introduce | transduced the amino group and the maleimide group on the silicon substrate. In Example 5, it is a figure showing the result of having immersed the solid support body in the EGFP solution of each density | concentration, immobilizing EGFP, and measuring the fluorescence. In Example 5, it is a figure showing the result of having reacted the primary antibody and the secondary antibody after GFP immobilization, and having detected by chemiluminescence.

SEQ ID NOs: 1-2 Synthetic DNA
SEQ ID NOS: 3-13 Synthetic peptides

Claims (14)

Formula I on the surface of the substrate:

[Wherein R is a divalent hydrocarbon group having 1 to 12 chain members]
A method for immobilizing a polypeptide by binding a polypeptide having an oligocysteine sequence introduced thereto to a maleimide group of a solid support having a group represented by the formula:   The method of claim 1, wherein the substrate further comprises a carbon layer. Formula I on the surface of the substrate:

[Wherein R is a divalent hydrocarbon group having 1 to 12 chain members]
A polypeptide-immobilized solid support, wherein a polypeptide into which an oligocysteine sequence is introduced is bound to a maleimide group of the solid support having a group represented by   The polypeptide-immobilized solid support according to claim 3, wherein the substrate further has a carbon layer.   The polypeptide-immobilized solid support according to claim 3 or 4, wherein an oligohistidine sequence is further introduced into the polypeptide into which the oligocysteine sequence has been introduced.   A method for detecting an interaction between an immobilized polypeptide and a sample polypeptide by bringing the sample polypeptide into contact with the polypeptide-immobilized solid support according to any one of claims 3 to 5.   7. The method of claim 6, wherein the interaction is detected by labeling the sample polypeptide and detecting a label derived from the sample polypeptide that has interacted with the immobilized polypeptide.   8. The method of claim 7, wherein the interaction of the polypeptide is detected using chemiluminescence. An oligocysteine sequence is introduced into the polypeptide, and the polypeptide into which the oligocysteine sequence has been introduced is represented by the formula I:

[Wherein R is a divalent hydrocarbon group having 1 to 12 chain members]
A method for purifying a polypeptide by reacting with a maleimide group on a solid support having a group represented by the following formula, and binding the polypeptide introduced with an oligocysteine sequence onto the solid support.   The method of claim 9, wherein the substrate further comprises a carbon layer. Formula I on the surface of the substrate:

[Wherein R is a divalent hydrocarbon group having 1 to 12 chain members]
A solid support for immobilizing a polypeptide having a group represented by the formula:   The solid support according to claim 11, wherein the substrate further comprises a carbon layer.   A vector for producing a gene encoding a polypeptide into which an oligocysteine sequence and an oligohistidine sequence are introduced.   A polypeptide into which an oligocysteine sequence and an oligohistidine sequence have been introduced.

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