JPWO2008081938A1 - Utilization of thermostable biotin-binding protein and solid support to which the protein is bound - Google Patents

Abstract

The present invention relates to a solid support to which a thermostable biotin-binding protein is linked. The present invention also relates to the use of a solid support to which the thermostable biotin-binding protein of the present invention is linked. The present invention further relates to technical fields such as purification, concentration, detection, and capture of a substance linked to biotin using a thermostable biotin-binding protein. Since the biotin-binding protein used for the solid support of the present invention has heat resistance, it is useful for use in an assay system involving exposure to a temperature of 70 ° C. or higher.

Description

  The present invention relates to a solid support to which a thermostable biotin-binding protein is linked. The invention also relates to the use of the solid support of the invention. The present invention further relates to technical fields such as purification, concentration, detection, and capture of a substance linked to biotin using a thermostable biotin-binding protein.

The affinity between avidin and biotin, or between streptavidin and biotin is very high (Kd = 10 ⁻¹⁵ to ⁻¹⁴ M), and is one of the strongest interactions as a bimolecular bimolecular interaction. Currently, the avidin / streptavidin-biotin interaction is widely applied in the fields of biochemistry, molecular biology, and medicine (Green, (1975), Adv. Protein Chem., 29: 85-133; Green, (1990), Methods Enzymol., 184: 51-67). Avidin is a basic glycoprotein derived from egg white and has an isoelectric point exceeding 10. Avidin has a problem of non-specific binding to DNA or the like due to its high basicity or sugar chain, which is a limiting factor in the use of avidin. On the other hand, streptavidin is derived from Streptomyces avidinii, and its isoelectric point is neutral and does not contain sugar chains. Both proteins form a tetramer and bind to one molecule of biotin per subunit. The molecular weight is about 60 kDa.

  Tamavidin (Tamavidin 1: SEQ ID NO: 2) is a rice blast fungus M. pneumoniae. As a protein exhibiting antibacterial activity against grisea, it is a third biotin-binding protein purified from edible mushrooms, and its gene structure has also been revealed (WO 02/072817). The homologue (tamavidin 2: SEQ ID NO: 4) was also identified from the same mushroom and successfully produced a recombinant protein (WO 02/072817). Tamavidin homologues can be easily produced by E. coli expression and purification using an iminobiotin column, which is a significant advantage compared to avidin and streptavidin production systems.

  When avidin is expressed in E. coli, the yield of soluble protein is as low as 50 μg per 50 ml (Airenne et al., 1994, Gene, 144: 75-80). Therefore, insect cell systems using baculovirus are now used (Airenne et al., 1997, Protein exp. Purif., 9: 100-108). When streptavidin is expressed in E. coli, the recombinant protein forms an insoluble inclusion body. This inclusion body is solubilized with a high concentration of guanidine hydrochloride, and then the protein is refolded by gradual removal of guanidine hydrochloride by dialysis to obtain soluble and active recombinant streptavidin (Sano and Cantor, 1990). , Proc. Natl. Acad. Sci. USA, 87: 142-146). Thus, production of avidin and streptavidin requires a lot of labor and time. On the other hand, when tamavidin homologue was expressed in E. coli, 1 mg of recombinant protein was obtained per 50 ml of culture. This is a high value for the production efficiency of biotin-binding protein, indicating the potential usefulness of tamavidin homologue protein.

So far, in the field of reagents and diagnostic agents, solid carriers to which avidin or streptavidin is bound, such as magnetic beads, microplates, sensor chips, etc., have been developed. No one having stability at high temperatures has been reported yet.
International Publication No. WO 02/072817 Pamphlet Green, 1975, Adv. Protein Chem., 29: 85-133 Green, 1990, Methods Enzymol., 184: 51-67 Airenne et al., 1994, Gene, 144: 75-80 Airenne et al., 1997, Protein exp. Purif., 9: 100-108 Sano and Cantor, 1990, Proc. Natl. Acad. Sci. USA, 87: 142-146

  An object of the present invention is to provide a solid support to which a thermostable biotin-binding protein is linked. Another object of the present invention is to provide a method for purification, concentration, detection, capture, etc. of a substance linked to biotin using a thermostable biotin-binding protein. In so doing, the use of the solid support of the present invention is provided. Another object of the present invention is to provide a solid support linked with a biotin-binding protein that can be used in an assay system involving exposure to a high temperature of 70 ° C. or higher.

  “Tamavidin” is a biotin-binding protein derived from the basidiomycete Tamogitake, and there are two types of tamavidin 1 and tamavidin 2 (see WO 02/0772817). As a result of intensive studies, the present inventors have clarified that tamavidin 2 has a very high affinity for biotin, like conventional avidin and streptavidin. That is, it was clarified that tamavidin 2 and biotin have affinity about 1000 times stronger than many antigen-antibody reactions. In addition, it has been demonstrated that there is almost no non-specific binding (to DNA), which is a problem in conventional avidin. Furthermore, tamavidin 2 has been found to have a heat resistance of 10 ° C. or more higher than that of streptavidin, and it has been found that this property is maintained even after tamavidin 2 is bound to a solid support. Furthermore, as a result of intensive studies, the present inventors have found that tamavidin 1 binds strongly to biotin and is more heat resistant at 5 ° C. than streptavidin. As a result of these studies, the present inventors have found that it is possible to provide a solid support to which a thermostable biotin-binding protein is linked, which retains the biotin-binding activity even under high temperature conditions. did. Therefore, the present invention provides a solid support to which a thermostable biotin-binding protein is linked, its use, and a method for purification, concentration, detection and capture of a substance linked to biotin using the thermostable biotin-binding protein. provide.

  Hereinafter, the present invention will be described in detail.

Solid carrier to which thermostable biotin-binding protein is linked The present invention provides a solid carrier to which thermostable biotin-binding protein is linked.

  In the present specification, the thermostable biotin-binding protein means tamavidin 1, tamavidin 2, or a variant thereof. Specifically, the thermostable biotin-binding protein linked to the solid support of the present invention is a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or the base sequence of SEQ ID NO: 1 or SEQ ID NO: 3. It may be a protein encoded by the nucleic acid comprising. Alternatively, the thermostable biotin-binding protein linked to the solid support of the present invention comprises a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or a base sequence of SEQ ID NO: 1 or SEQ ID NO: 3. It is a variant of a protein encoded by a nucleic acid, and may be a protein having biotin-binding activity and heat resistance similar to tamavidin 1 or 2. In this specification, tamavidin 1, tamavidin 2, and variants thereof may be collectively referred to simply as tamavidin.

  A tamavidin 1 or 2 variant is a protein comprising an amino acid sequence comprising a deletion, substitution, insertion and / or addition of one or more amino acids in the amino acid sequence of SEQ ID NO: 2 or 4, Alternatively, it may be a protein having biotin-binding activity and heat resistance similar to 2. The substitution may be a conservative substitution, which is the replacement of a particular amino acid residue with a residue having similar physicochemical characteristics. Non-limiting examples of conservative substitutions include substitutions between aliphatic group-containing amino acid residues such as Ile, Val, Leu or Ala mutual substitutions, Lys and Arg, Glu and Asp, Gln and Asn mutual substitutions. Substitution between polar residues such as substitution is included.

  Mutations resulting from amino acid deletions, substitutions, insertions and / or additions may be performed on the DNA encoding the wild-type protein, for example by site-directed mutagenesis (eg, Nucleic Acid Research, Vol. 10, No. 20), which is a well-known technique. , p. 6487-6500, 1982, the entire contents of which are incorporated herein by reference). As used herein, “one or more amino acids” means amino acids that can be deleted, substituted, inserted and / or added by site-directed mutagenesis. In the present specification, “one or more amino acids” may mean one or several amino acids depending on the case.

  Site-directed mutagenesis can be performed, for example, using a synthetic oligonucleotide primer complementary to the single-stranded phage DNA to be mutated, in addition to the specific mismatch that is the desired mutation, as follows: . That is, the synthetic oligonucleotide is used as a primer to synthesize a strand complementary to the phage, and a host cell is transformed with the obtained double-stranded DNA. Transformed bacterial cultures are plated on agar and plaques are formed from single cells containing phage. Then, theoretically 50% of the new colonies contain phages with mutations as single strands and the remaining 50% have the original sequence. The obtained plaque is hybridized with a synthetic probe labeled by kinase treatment at a temperature that hybridizes with the DNA having the desired mutation and that does not hybridize with DNA having the original strand. . Next, plaques that hybridize with the probe are picked up and cultured to recover DNA.

  In addition to the above-mentioned site-directed mutagenesis, a method for performing deletion, substitution, insertion and / or addition of one or more amino acids while maintaining the activity of the amino acid sequence of a biologically active peptide There are also methods of treating the gene with a mutagen and methods of selectively cleaving the gene, then removing, substituting, inserting or adding selected nucleotides and then ligating.

  The tamavidin 1 or 2 variant further has at least 60% or more, preferably 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, with the amino acid sequence of SEQ ID NO: 2 or 4. A protein comprising an amino acid sequence having an amino acid identity of 95% or more, 98% or more, or 99% or more, more preferably 99.3% or more, and has the same biotin-binding activity and heat resistance as tamavidin 1 or 2 It may be a protein having

  The percent identity between two amino acid sequences may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences is based on the algorithm of Needleman, SB and Wunsch, CD (J. Mol. Biol., 48: 443-453, 1970), and the University of Wisconsin Genetics Computer Group (UWGCG) It may be determined by comparing the sequence information using a more available GAP computer program. Preferred default parameters for the GAP program include: (1) Scoring matrix as described in Henikoff, S. and Henikoff, JG (Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992). , Blosum62; (2) 12 gap weights; (3) 4 gap length weights; and (4) no penalty for end gaps.

  Other programs used by those skilled in the art of sequence comparison may also be used. The percent identity can be determined by comparison with sequence information using, for example, the BLAST program described in Altschul et al. (Nucl. Acids. Res., 25, p. 3389-3402, 1997). The program can be used on the Internet from the National Center for Biotechnology Information (NCBI) or the DNA Data Bank of Japan (DDBJ) website. Various conditions (parameters) for identity search by the BLAST program are described in detail on the same site, and some settings can be changed as appropriate, but the search is usually performed using default values. Alternatively, the percent identity between two amino acid sequences can be determined by genetic information processing software GENETYX Ver. The program may be determined using a program such as 7 (manufactured by Genetics) or the FASTA algorithm. At that time, the default value may be used for the search.

  The percent identity between two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or more preferably, this comparison is made by comparing the sequence information using a computer program. A typical preferred computer program is the Wisconsin package, version 10.0 program “GAP” from the Genetics Computer Group (GCG; Madison, Wis.) (Devereux, et al., 1984, Nucl. Acids Res ., 12: 387). By using this “GAP” program, in addition to comparing two nucleic acid sequences, two amino acid sequences can be compared, and a nucleic acid sequence and an amino acid sequence can be compared. Here, the preferred default parameters for the “GAP” program are: (1) GCG run of unary comparison matrix for nucleotides (including values of 1 for identical and 0 for non-identical), Schwartz and Gribskov and Burgess, Nucl. Acids Res., 14 as described by Dayhoff, “Atlas of Polypeptide Sequence and Structure,” National Biomedical Research Foundation, pages 353-358, 1979. : Weighted amino acid comparison matrix of 6745, 1986; or other comparable comparison matrix; (2) 30 penalties for each gap of amino acids and one additional penalty for each symbol in each gap; or each gap in nucleotide sequence About 50 penalties and an additional for each symbol in each gap 3 penalties; (3) no penalty to end gaps; and (4) no maximum penalty for long gaps. Other sequence comparison programs used by those skilled in the art are available, for example, at the National Library of Medicine website: http://www.ncbi.nlm.nih.gov/blast/bl2seq/bls.html A BLASTN program, version 2.2.7, or UW-BLAST 2.0 algorithm can be used. Standard default parameter settings for UW-BLAST 2.0 are described at the following Internet site: http://blast.wustl.edu. In addition, the BLAST algorithm uses a BLOSUM62 amino acid scoring matrix and the selection parameters that can be used are: (A) a segment of query sequence with low composition complexity (Wootton and Federhen's SEG program (Computers and Chemistry, 1993); Wootton and Federhen, 1996 “Analysis of compositionally biased regions in sequence databases” Methods Enzymol., 266: 544-71) Or include a filter to mask segments consisting of short-periodic internal repeats (determined by the XNU program of Claverie and States (Computers and Chemistry, 1993)), and (B) match against database sequences A statistical significance threshold to report, or According to the statistical model of the E-score (Karlin and Altschul, 1990), the expected probability of a match found by chance; if the statistically significant difference due to a match is greater than the E-score threshold, the fit is Not reported); the preferred E-score threshold value is 0.5 or, in order of increasing preference, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001 1e-5, 1e-10, 1e-15, 1e-20, 1e-25, 1e-30, 1e-40, 1e-50, 1e-75, or 1e-100.

  A tamavidin 1 or 2 variant is also a protein encoded by a nucleic acid comprising a base sequence that hybridizes under stringent conditions to the complementary strand of the base sequence of SEQ ID NO: 1 or 3, comprising a tamavidin 1 or 2 It may be a protein having the same biotin-binding activity and heat resistance.

  Here, “under stringent conditions” means to hybridize under moderately or highly stringent conditions. Specifically, moderately stringent conditions can be easily determined by those skilled in the art having general techniques based on, for example, the length of the DNA. Basic conditions are shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Chapters 6-7, Cold Spring Harbor Laboratory Press, 2001, and for nitrocellulose filters, 5 × SSC, 0.5 Pre-wash solution of% SDS, 1.0 mM EDTA, pH 8.0, about 50% formamide at about 40-50 ° C., 2 × SSC-6 × SSC (or about 50% formamide at about 42 ° C. , Other similar hybridization solutions such as Stark's solution) and the use of, for example, about 40 ° C-60 ° C, 0.5-6 x SSC, 0.1% SDS wash conditions Is included. Preferably, moderately stringent conditions include hybridization conditions (and wash conditions) at about 50 ° C. and 6 × SSC. High stringency conditions can also be readily determined by one skilled in the art based on, for example, the length of the DNA.

In general, these conditions include hybridization at higher temperatures and / or lower salt concentrations than moderately stringent conditions (eg, about 65 ° C., 6 × SSC to 0.2 × SSC, preferably 6 × SSC, More preferably 2 × SSC, most preferably 0.2 × SSC hybridization) and / or washing, eg hybridization conditions as described above, and approximately 65 ° C.-68 ° C., 0.2 × SSC, 0 Defined with 1% SDS wash. In hybridization and wash buffers, SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate) to SSPE (1 × SSPE is 0.15 M NaCl, 10 mM NaH ₂ PO ₄ , and 1. 25 mM EDTA, pH 7.4) can be substituted, and washing is performed for 15 minutes after hybridization is complete.

  In addition, a commercially available hybridization kit that does not use a radioactive substance for the probe can also be used. Specific examples include hybridization using an ECL direct labeling & detection system (manufactured by Amersham). For stringent hybridization, for example, 5% (w / v) Blocking reagent and 0.5M NaCl are added to the hybridization buffer in the kit, and the reaction is performed at 42 ° C. for 4 hours. A condition is that 20% is performed twice at 55 ° C. for 20 minutes in 4% SDS, 0.5 × SSC, and once at room temperature for 5 minutes in 2 × SSC.

  The biotin-binding activity and heat resistance of the tamavidin 1 or 2 mutant can be measured by any known method. For example, it may be measured by a method using fluorescent biotin as described in Kada et al. (Biochim. Biophys. Acta., 1427: 44-48 (1999)). This method is an assay system utilizing the property that the fluorescence intensity of fluorescent biotin disappears when fluorescent biotin binds to the biotin binding site of the biotin binding protein. Alternatively, the biotin-binding activity of the mutant protein can be evaluated using a sensor capable of measuring the binding between the protein and biotin, such as a biosensor based on surface plasmon resonance. The heat resistance can be evaluated by changing the measurement temperature when measuring the above biotin binding activity.

The heat-resistant biotin-binding protein linked to the solid support of the present invention has a high affinity for biotin, and its dissociation constant K _d is on the order of 10 ⁻⁸ M or less, preferably 10 ⁻⁹ M. Below the order, below the order of 10 ⁻¹⁰ M, below the order of 10 ⁻¹¹ M, below the order of 10 ⁻¹² M, below the order of 10 ⁻¹³ M. Typically, the dissociation constant K _d may be on the order of 10 ⁻¹³ to 10 ⁻⁹ M.

  The heat-resistant biotin-binding protein linked to the solid support of the present invention has higher heat resistance than known egg white-derived avidin or streptavidin. Here, heat resistance refers to both protein stability in a high temperature range and biotin binding activity in a high temperature range.

  The protein stability in the high temperature range can be evaluated as a temperature at which the color development of the protein band disappears by 50% in SDS-PAGE analysis as compared with the case of room temperature. For the thermostable biotin-binding protein linked to the solid support of the present invention, the temperature at which the color development of the protein band disappears by 50% compared to the case of room temperature in SDS-PAGE analysis is higher than 71 ° C., preferably 75 ° C. or higher. 77.5 ° C. or higher, 80 ° C. or higher, 82.5 ° C. or higher, 85 ° C. or higher, 87 ° C. or higher.

  The biotin binding activity in the high temperature range can be evaluated as a temperature at which biotin binding is reduced by 50% compared to the case of room temperature. For the thermostable protein linked to the solid support of the present invention, the temperature at which the binding of biotin is reduced by 50% compared to that at room temperature is higher than 73 ° C, preferably 75 ° C or higher, 78 ° C or higher, 80 ° C or higher, It may be 82.5 ° C. or higher and 85 ° C. or higher.

  The thermostable biotin-binding protein linked to the solid support of the present invention can be obtained by purifying a protein derived from basidiomycetes or as a recombinant protein.

  In the present invention, the solid carrier to which the thermostable biotin-binding protein is linked is particularly limited as long as it is a solid or insoluble material (for example, a material that can be separated from the reaction mixture by filtration, precipitation, magnetic separation, etc.). Not.

The material constituting the solid carrier include cellulose, Teflon ^TM, nitrocellulose, agarose, dextran, chitosan, polystyrene, polyacrylamide, polyester, polycarbonate, polyamide, polypropylene, nylon, polydiene vinylidene difluoride, latex, silica, glass, glass These include fiber, gold, platinum, silver, copper, iron, stainless steel, ferrite, silicon wafer, polyethylene, polyethyleneimine, polylactic acid, resin, polysaccharide, protein (such as albumin), carbon or combinations thereof, etc. It is not limited to.

  The shape of the solid support includes, but is not limited to, beads, magnetic beads, thin films, microtubes, filters, plates, microplates, carbon nanotubes, sensor chips, and the like. Flat solid carriers such as thin films and plates may be provided with pits, grooves, filter bottoms, etc., as is known in the art.

  In one aspect of the invention, the magnetic beads can have a sphere diameter ranging from about 25 nm to about 1 mm. In preferred embodiments, the magnetic beads have a diameter in the range of about 50 nm to about 10 μm. The size of the magnetic beads can be selected depending on the particular application. Because some bacterial spores have a size on the order of about 1 μm, preferred beads for capturing such spores have a diameter greater than 1 μm.

  In one aspect of the invention, beads composed of highly cross-linked spherical agarose, such as Sepharose, can have a diameter in the range of about 24 μm to about 165 μm. In preferred embodiments, the highly crosslinked spherical agarose beads have a diameter in the range of about 24 μm to about 44 μm. The size of the highly crosslinked spherical agarose beads can be selected depending on the particular application.

  Examples of solid supports having a hydrophobic surface include polystyrene latex beads such as those commercially available from Polysciences, Warrington, PA or Spherotech, Liberville, IL.

Silica (SiO ₂₎ - Examples of processing or silica (SiO ₂₎ based solid carrier, Polysciences, Warrington, available from PA, include superparamagnetic silica beads or the like, which, nucleic acid (e.g., DNA) Can be used to capture. Alternatively, M-280 or the like commercially available from Dynal Biotech can also be used.

  Magnetic beads having a hydrophilic surface can be used to capture growing bacterial cells, nucleic acids and other components. Examples of such magnetic beads include beads commercially available from Polysciences, Warrington, PA under the name Biomag® carboxyl or beads having the name MC02N / 2928 under Bangs Laboratory, Inc., Fishers, IN. Alternatively, M-270 or the like commercially available from Dynal Biotech can be used.

  The thermostable biotin-binding protein and the solid support can be linked using a protein-solid support coupling method known to those skilled in the art. For example, the surface of the solid support is modified so that the carboxyl group is exposed, and the carboxyl group and the amino group of the protein are converted in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) which is a crosslinking reagent. A protein and a solid support can be linked by a coupling reaction. Alternatively, for example, by mixing a solid carrier in which the carboxyl group on the surface of the solid carrier is active esterified with N-hydroxysuccinimide (NHS) and a protein in a pH 6.5 to 9 buffer solution containing no primary amino group. The carboxyl group on the surface of the solid support and the amino group of the protein can be linked.

  Alternatively, by using the crosslinking reagent BS3 (bis [sulfosuccinimidyl] suberate) or DSS (disuccinimidyl suberate), the amino group on the surface of the solid support and the amino group of the protein, or the crosslinking reagent SPDP (N- Using succinimidyl 3- [2-pyridyldithio] propionate) or GMBS (N- (4-maleimidobutyryloxy) succinimide), the amino group on the surface of the solid support and the thiol group of the protein can be linked.

  The thermostable biotin-binding protein linked to the solid support of the present invention retains protein stability and biotin-binding property at high temperatures. For this reason, the solid carrier of the present invention can be treated at a temperature higher than that of streptavidin, which has been most used in the past, while maintaining the biotin binding ability. The solid carrier of the present invention is 70 ° C to 90 ° C, preferably 75 ° C to 90 ° C, 80 ° C to 90 ° C, 85 ° C to 90 ° C, 70 ° C to 85 ° C, 70 ° C to 80 ° C, 75 ° C to 80 ° C. Even when subjected to exposure treatment under heating conditions of 75 ° C. to 85 ° C., 75 ° C. to 87.5 ° C., the biotin-binding ability can be maintained. This means that, for example, nucleic acid hybridization at a higher temperature and subsequent washing can be performed, that is, hybridization with higher stringency becomes possible. By using the solid support of the present invention, it is possible to construct an assay system capable of suppressing nonspecific hybridization as much as possible. Alternatively, for example, when tamavidin is linked to a solid support, it is possible to select a method that requires high-temperature treatment. Furthermore, when binding heat-resistant protein, enzyme, fluorescent dye or the like to tamavidin, or when reacting with a biotin modification product after binding, it is possible to cause a more specific reaction using high temperature.

  In addition, since the thermostable biotin-binding protein linked to the solid support of the present invention has almost no non-specific binding to DNA, non-specific binding to DNA which is a problem in egg white-derived avidin known in the art. This problem can also be avoided.

Separation, concentration, purification, detection and / or capture method of a substance linked to biotin using a thermostable biotin-binding protein The present invention relates to separation, concentration, capture, purification and / or detection of a substance linked to biotin. A method comprising the following steps:
1) A solid carrier linked to tamavidin of the present invention and a substance linked to biotin are brought into contact with each other, and the substance linked to biotin is bound to the solid carrier;
2) washing away contaminants not bound to the solid support; and 3) separating, concentrating, capturing or purifying the material by recovering the material linked to biotin bound to the solid support, and / or Detect the substance;
The method is provided comprising: In a preferred embodiment, at least one of the above-described steps in the method of the present invention is performed under heating conditions of 70 ° C. to 90 ° C., more preferably 75 ° C. to 90 ° C., 80 ° C. to 90 ° C., 85 ° C. to 90 ° C. , 70 ° C to 85 ° C, 70 ° C to 80 ° C, 75 ° C to 80 ° C, 75 ° C to 85 ° C, 75 ° C to 87.5 ° C.

  In the method of the present invention, a substance linked to biotin refers to both a substance linked directly or indirectly to biotin. Direct linkage of biotin and substance is achieved by covalent linkage. Indirect linking of biotin and substance is achieved by further linking the substance to a ligand covalently linked to biotin by covalent bond, ionic bond, hydrogen bond, or hydrophobic interaction. Specific examples of indirect ligation include ligation of antigen molecules by antigen-antibody reaction when using biotinylated antibodies, and ligation of complementary nucleic acids by hybridization of nucleic acids when using biotinylated nucleic acid probes. Can be mentioned.

  In a preferred embodiment, at least one step in the method of the present invention is performed under heating conditions of 70 ° C. to 90 ° C., so that indirect linking of biotin and substance is performed under the temperature conditions used in the method of the present invention. Is preferably retained. For example, when nucleic acid hybridization is used as an indirect link between biotin and a substance, the length of the nucleotide to hybridize is at least 20 nucleotides or more, preferably at least 25 nucleotides or more, 30 nucleotides or more, 35 nucleotides or more, It may be 40 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more.

  The material constituting the solid support to which tamavidin is linked and the shape thereof used in the method of the present invention can be selected according to the characteristics of the substance to be separated, concentrated, purified, detected and / or captured.

  In addition, the composition of the buffer used in each step of the method of the present invention, the temperature to be applied, and the like can be appropriately set by those skilled in the art in consideration of the properties of the substance to be separated. A method for detecting a substance separated, concentrated, captured and / or purified by the method of the present invention can be appropriately selected by those skilled in the art depending on the properties of the substance.

  The method of the present invention is not limited thereto, but includes, for example, detection of nucleic acids (Japanese Patent Application Laid-Open No. 2003-125800), apparatus and method for isolating nucleic acids from a sample (Boutlin et al., WO 2004/005553), Nucleic acid amplification method: Method according to hybridization signal amplification method (HSAM) (Zan et al., WO 1998/004745) or virus and virus genome concentration (Tamatsukuri, 2004, BIO INDUSTRY, 21 (8): 39-47) It may be. Alternatively, it can be used for detection, capture and concentration of cells and microorganisms.

  For example, when concentrating a virus in a body fluid, first, a specimen is incubated with a biotinylated antibody that specifically binds to a virus surface antigen in a suitable buffer solution. The biotinylation of the antibody can be performed using, for example, a kit commercially available from Pierce et al. Next, a solid support to which the tamavidin of the present invention is linked, for example, magnetic beads, is added and mixed. Finally, using a magnet, the virus-antibody-biotin-tamavidin-magnetic bead complex is aggregated, the supernatant is removed, washed several times with an appropriate buffer, the magnet is removed, and the desired Suspend in buffer to complete virus concentration.

  Alternatively, when concentrating viral genomic DNA in a body fluid, first, a specimen is placed in an appropriate solution that destroys viral particles, and genomic DNA is extracted from the virus. If necessary, a step for single-stranding this genome is included. Subsequently, a biotinylated single-stranded oligo DNA complementary to several tens to several hundred bases complementary to a part of the viral genome, or two tens to several hundred bases having a complementary chain to a part of the viral genome. The strand DNA is incubated with biotinylated and heat denatured, and the biotinylated oligo DNA and the virus genome are hybridized. When using the solid carrier linked with tamavidin of the present invention, the temperature is higher than when using a solid carrier linked with conventional avidin or streptavidin, for example, 70 ° C. to 90 ° C., preferably 75 ° C. to 90 ° C., 80 ° C. Temperature ranges of 90 ° C, 85 ° C to 90 ° C, 70 ° C to 85 ° C, 70 ° C to 80 ° C, or 75 ° C to 80 ° C, 75 ° C to 85 ° C, 75 ° C to 87.5 ° C are applicable. is there. By applying such a high temperature to the hybridization temperature, binding of non-specific DNA is suppressed, and DNA with a higher specificity can be concentrated without DNA other than the desired viral genome being mixed as much as possible. It becomes. Next, a solid support to which the tamavidin of the present invention is linked, for example, magnetic beads, is added to a sample in a high temperature state (a state where the biotinylated oligo DNA specifically captures the virus genome) and mixed. Finally, using a magnet, the virus genome-oligo DNA-biotin-tamavidin-magnetic bead complex is aggregated, the supernatant is removed, and after washing several times with an appropriate buffer, the magnet is removed, Suspend in the desired buffer to complete the concentration of the viral genome. Thereafter, detection of the viral genome can be performed using, for example, PCR (Saiki et al. (1985) Science 230: 1350-1354). Tamavidin has higher heat resistance in the absence of biotin than conventional avidin and streptavidin, and has almost no non-specific binding to DNA. Therefore, for example, it is suitable for specific concentration of DNA by the method as described above.

  Tamavidin linked to the solid support of the present invention retains protein stability and biotin binding at high temperatures. For this reason, the solid carrier of the present invention can be treated at a temperature higher than that of streptavidin, which has been most used in the past, while maintaining the biotin binding ability. By using the solid support of the present invention, it is possible to construct an assay system including treatment in a high temperature range.

FIG. 1 is a photograph (A) and a graph (B) showing the results of a non-specific binding test for tamavidin 2, streptavidin and avidin to DNA. FIG. 2 shows the results of evaluating the heat resistance (protein stability) of tamavidin 2 compared to streptavidin. FIG. 2A shows the result of SDS-PAGE, and FIG. 2B is a graph showing the quantification result of the protein band. FIG. 3 is a graph showing the heat resistance (fluorescence biotin binding activity) of tamavidin 2 (A), streptavidin (B) and avidin (C). FIG. 4 is a graph showing the heat resistance (fluorescence biotin binding activity) of tamavidin 2 magnetic beads (A), commercially available streptavidin magnetic beads (B), streptavidin magnetic beads (C), and avidin magnetic beads (D). is there. FIG. 5 is a graph showing heat resistance (fluorescence biotin binding activity) of tamavidin 2 magnetic beads (A and C) and commercially available streptavidin magnetic beads (B and D). FIG. 6 is a graph showing the heat resistance (fluorescence biotin binding activity) of tamavidin 2 Sepharose beads. FIG. 7 is a graph showing the fluorescent biotin binding activity of tamavidin 1.

Example 1: Tamavidin 2 (TM2)
1-1. Characterization of tamavidin 2
Expression and purification of tamavidin 2 in E. coli When DNA (SEQ ID NO: 3) encoding tamavidin 2 (TM2) is incorporated into the expression vector pTrc99A and expressed in E. coli, most of the expressed TM2 accumulates in the soluble fraction and the expression level (WO 02/072817). TM2 protein was purified from recombinant E. coli using a column packed with iminobiotin agarose (Sigma) according to the method of Hofmann et al. (Proc. Natl. Acad. Sci. USA, 77: 4666-4668, 1980). Specifically, E. coli described in WO 02/072817 was subjected to expression induction at 1 mM IPTG at 37 ° C. for 5 hours and then collected, and the pellet was suspended in 50 mM Caps pH 11.0, 50 mM NaCl. The cells were destroyed by ultrasound. The supernatant after centrifugation was applied to a self-made iminobiotin column (height 3 cm, volume 0.5 ml) equilibrated with 50 mM Caps pH 11.0, 50 mM NaCl. After washing with 5 ml of 50 mM Caps pH 11.0, 500 mM NaCl, elution was performed with 1.5-2 ml of 50 mM NH ₄ OAc pH 4.0. The yield of purified protein was approximately 1 mg from 50 mL culture.

Mass spectrometry tamavidin 2 was dissolved in 0.1% TFA, 50% MeCN saturated solution at a concentration of 10 mg / ml, and this sample solution was purified with ZipTip C4 (Millipore) and directly applied to a MALDI plate. After air drying, a matrix solution (sinapic acid) was overlaid. Further, after air drying, the sample was mounted on a laser ionization time-of-flight mass spectrometer (MALDI-TOFMS) AXIMA-CFR (Shimadzu Corporation) and subjected to mass spectrometry (drawing voltage: 20 kV, flight mode: Linear, detected ion: Positive). As a result, m / z = 15146.3 (corresponding to the monomer), 30335.0 (corresponding to the dimer), and 60932.0 (corresponding to the tetramer) were observed. Furthermore, in the case of biotin-conjugated tamavidin 2, the peak corresponding to the tetramer was large. From this result, tamavidin 2 was determined to be a tetramer and a mass of 60932. Analysis of the N-terminal sequence of the protein revealed that the serine next to the translation initiation methionine is the N-terminus. Genetic information processing software GENETYX Ver. Using 7 (manufactured by Genetics), the isoelectric point of a polypeptide consisting of 140 amino acids excluding the starting methionine of tamavidin 2 was 7.36.

Molecular Absorption Coefficient The molecular extinction coefficient of tamavidin 2 is A ₂₈₀ = 41750 M ⁻¹ cm ⁻¹ subunit ⁻¹ (0.68 at 0.25 mg / mL) as a theoretical value. A sample obtained by actually purifying tamavidin 2 and dialyzing it against ammonium acetate was freeze-dried (ammonium acetate was fully sublimated) and weighed 3 mg. When this sample was dissolved in 20 mM KPi (pH 6.5), the concentration was adjusted to 0.25 mg / mL, and A ₂₈₀ was measured, an actual measurement value of 0.67 was obtained. This corresponded to 98% of the theoretical value. Thus, it has become possible to easily measure the tamavidin concentration by measuring the A _280.

Measurement of activity with fluorescent biotin Biotin binding activity of tamavidin 2 with fluorescent biotin was measured according to the method of Kada et al. (Biochim. Biophys. Acta., 1427: 44-48, (1999)). 200μL assay buffer in _{(50mM NaH 2 PO 4, 100mM} NaCl, 1mM EDTA (pH7.5)), and purified TM2 was adjusted so as to be included in the stepwise from 0pmol to 486Pmol. To this solution, 50 μL (1 nmol) of 20 pmol / μL fluorescent biotin solution (biotin-4-fluorescein: Molecular Probe) was mixed, allowed to stand at room temperature for 10 minutes, and then fluorescence intensity was measured using Las-3000 (FUJIFILM). As a result, 0.274 nmol TM2 was bound to 1 nmol fluorescent biotin. That is, 3.6 mol of fluorescent biotin was bound to 1 mol of tamavidin. This showed that 4 molecules of fluorescent biotin (1 molecule per subunit) bind to 1 molecule of tamavidin. Similarly, streptavidin bound to 3.4 mol of fluorescent biotin with respect to 1 mol.

1-2. Nonspecific binding of tamavidin 2 (TM2)
Purification of Rabbit Anti-TM2 Antibody Two types of antibodies were prepared by using tamavidin 2 (TM2) protein expressed in E. coli with an iminobiotin column and further gel-purifying it. The detection sensitivity by Western method using an alkaline phosphatase-labeled anti-IgG antibody was about 0.5 ng with respect to two purified recombinant tamavidins. From the above results, it was concluded that antibodies with high specificity and titer were completed. Although the anti-tamavidin 2 antibody-tamavidin 1 cross-reactivity was low, it was detected (about 1/20 of the original antigen).

  Anti-TM2 antibody (an antibody prepared from an antigen subjected to only iminobiotin column purification) was further purified as follows. 40 μg of TM2 was separated by SDS-PAGE using two 15% acrylamide gels, and the protein was transferred to two nitrocellulose membranes (BIO-RAD). Blocking was performed by shaking the membrane with TBS buffer containing 3% BSA for 1 hour at room temperature. Subsequently, after reacting with an anti-TM2 antibody (an antibody prepared from an antigen subjected to only iminobiotin column purification, diluted 1000-fold) overnight at room temperature, the site where TM2 is transferred is cut out, and an elution buffer ( The mixture was shaken in 0.2 M glycine, 1 mM EDTA pH 2.8) at room temperature for 20 minutes. After neutralization with 1/10 volume of 1M Tris solution of elution buffer, the same amount of 10 × TBS buffer was added and stored at 4 ° C.

Non-specific binding to DNA Non-specific binding test of tamavidin 2 (TM2) to DNA Non-specific binding of TM2 to DNA was analyzed. Salmon sperm DNA serially diluted with 2 × SSC buffer from 10 μg to 0.3 μg was alkali-denatured and adsorbed to Hybond N + membrane (Amersham Biosceinces) using Bio-Dot SF (BIO-RAD). After blocking the membrane with 5 × Denhardt's solution (0.1% BSA, 0.1% Ficoll, 0.1% polovinylprolidone), 25 μg / mL TM2, streptavidin, and avidin solution for 90 minutes at room temperature Soaked. Thereafter, the membrane was washed with TTBS buffer (TBS buffer containing 0.05% Tween 20) three times at room temperature for 5 minutes. The membrane was blocked with TBS buffer containing 0.5% skim milk and 0.01% Tween 20 for 1 hour. As a primary antibody, TM2 is a rabbit anti-TM2 antibody purified by the method described above, streptavidin is a rabbit anti-streptavidin antibody (SIGMA), avidin is a rabbit anti-avidin antibody (Abcam), and antibody titers are equivalent. It was diluted to be used. The antigen-antibody reaction with the primary antibody was performed overnight at room temperature. The membrane was washed three times for 5 minutes at room temperature with TTBS buffer, and then reacted with alkaline phosphatase-labeled anti-rabbit IgG antibody (BIO-RAD) (diluted 10,000 times) as the secondary antibody for 1 hour at room temperature. The membrane was washed three times for 5 minutes at room temperature with TTBS buffer, developed with Alkaline Phosphatase substrate kit II, vector Black (Funakoshi), and quantified with Las-3000 (FUJIFILM). As a result, the staining intensity of avidin increased depending on the DNA concentration, whereas the staining intensity of TM2 and streptavidin was not strongly influenced by the DNA concentration (FIGS. 1A and B). From these results, it was shown that tamavidin 2 has almost no non-specific activity with respect to DNA and is equivalent to streptavidin. This shows the superiority of tamavidin 2 over avidin.

1-3. Interaction analysis between tamavidin 2 and biotin
Kinetic analysis of the interaction between tamavidin 2 (TM2) and biotin using a Biacore biosensor 2 mg of highly purified BSA (Sigma) and 1 mg of NHS-biotin (Pierce) in 1 ml of 50 mM sodium borate pH 8.0 And incubated at 4 ° C. for 2 hours. NHS-biotin (EZ-Link NHS-LC-LC-Biotin) was added after dissolving in a small amount of DMSO in advance. This was placed in a dialysis tube (MWCO 6-8,000) and dialyzed overnight at 4 ° C. against 50 mM sodium carbonate pH 6.7. The biotin-BSA conjugate thus prepared (MW 67 kDa, 30 μM) was used as the ligand (substance to be attached to the sensor chip) of the Biacore (registered trademark) biosensor. On the other hand, recombinant tamavidin 2 was prepared as an analyte (substance flowing through the flow path system) and analyzed for molecular interactions by Biacore (registered trademark) 3000 (biosensor based on surface plasmon resonance, Biacore Inc.). went. Biotin-BSA and BSA as a negative control were immobilized on a CM5 sensor chip by an amine coupling method. The immobilization amount was adjusted to be about 200 RU. Chips with immobilized BSA were placed in flow cells 1 and 3, and chips with biotin-BSA solidified were placed in flow cells 2 and 4. Tamavidin 2 is in flow cells 1 and 2, and streptavidin is in flow cells 3 and 4 at a flow rate of 20 μl / min for 2 minutes, running buffer [10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactantat20 (Biacore Inc.)]. The sample dissociation was then monitored for 60 minutes. Since it was impossible to dissociate the bound tamavidin 2 and streptavidin, the regeneration operation was not performed in the measurement, and seven steps of measurement were performed from the low concentration side (3.125, 6.25, 12). .5, 25, 50, 100, and 200 nM). The BSA data was subtracted from the BSA-biotin data as a reference. The measurement was performed at 25 ° C. From the resulting sensorgram, using an analysis software BIAevaluation Ver. 4.1, 1: using 1 binding model performs kinetic analysis, the binding rate constant (k _a) and dissociation rate constants (k _d) Calculated. Dissociation constant (K _d) were calculated from k _d / k _a.

  In the case where the regeneration operation is not performed, Rmax (maximum amount of analyte binding) decreases each time measurement of each concentration is performed, but Rmax is locally fitted at the time of analysis and calculation is performed for each concentration. Moreover, only the data of the analyte concentration that can be approximated to the 1: 1 binding model was adopted.

  Table 1 shows the results of kinetic analysis of intermolecular interaction between recombinant tamavidin 2 and biotin by Biacore (registered trademark) 3000 (biosensor based on the principle of surface plasmon resonance).

得られたタマビジン２の解離定数は、１０^-12 Mのオーダーで、今回我々の測定したストレプトアビジンの解離定数と同じオーダーであった。この結果によって、タマビジン２は、アビジン、ストレプトアビジンに次ぐ、ビオチンと非常に高い親和性をもつタンパク質であることが明らかとなった。タマビジン２は、現在広く応用されているアビジン−ビオチン技術に応用が可能であると考えられる。

The dissociation constant of tamavidin 2 obtained was on the order of 10 ⁻¹² M, which was the same order as the dissociation constant of streptavidin we measured this time. From this result, it was revealed that tamavidin 2 is a protein having a very high affinity for biotin after avidin and streptavidin. It is considered that tamavidin 2 can be applied to avidin-biotin technology which is currently widely applied.

1-4. Heat resistance of tamavidin 2 The heat resistance of tamavidin 2 (TM2) was analyzed by comparison with streptavidin or avidin.

Protein Stability 0.2 μg / μL TM2 and 0.2 μg / μL Streptavidin were heated at room temperature, 50, 60, 70, 80, 90, 99 ° C. for 20 minutes at 10 μL (2 μg). Subsequently, the mixture was centrifuged at 15000 rpm for 10 minutes, and the soluble protein in the supernatant was added with an equal amount of 2 × SDS sample buffer (100 mM Tris-HCl pH 6.8, 12% 2-mercaptoethanol, 2% SDS, 20% glycerol). After suspending and heating at 95 ° C. for 10 minutes, SDS-PAGE was performed. The protein band was detected by CBB staining. A calibration curve was created based on a quantitative marker (LMW ELECTROPHORESIS CALIBRATION KIT; Pharmacia Biotech) using Las-3000 (FUJIFILM), and the protein band was quantified. SDS-PAGE results are shown in FIG. 2A, and protein band quantification results are shown in FIG. 2B. The temperature at which 50% of the protein disappeared was 71 ° C for streptavidin, whereas it was 87 ° C for tamavidin 2. As a result, it was revealed that tamavidin 2 has higher heat resistance by 15 ° C. or more than streptavidin. Moreover, when biotin was added to tamavidin 2, the heat resistance became extremely strong, and the tetramer was not dissociated even after treatment at 95 ° C.

Biotin binding activity Utilizing the property that fluorescent biotin loses its fluorescence intensity when fluorescent biotin is bound to the biotin binding site of biotin binding protein, the biotin binding ability of tamavidin 2 under high temperature conditions can be compared with streptavidin and avidin. Compared.

About 0.25 μg / μL of TM2 and streptavidin were heated at room temperature, 50, 60, 70, 80, and 90 ° C. for 20 minutes, and then 150 μL assay buffer (50 mM NaH ₂ PO ₄ , 100 mM NaCl, 1 mM EDTA (pH 7.5)). A solution containing stepwise TM2 or streptavidin from 0 μL to 27 μL was prepared. This solution was mixed with 50 μL (250 pmol) of a 5 pmol / μL fluorescent biotin solution (biotin-4-fluorescein: Molecular Probe), allowed to stand at room temperature for 20 minutes, and then measured for fluorescence intensity using Las-3000 (FUJIFILM).

  The heat resistance test of the biotin-binding protein was performed as follows. About 0.25 μg / μL of biotin-binding protein is heated at room temperature, 50, 60, 70, 80, and 90 ° C. for 20 minutes, and then stepped from 0 μL to 12 μL of heated biotin-binding protein in 150 μL assay buffer. Prepared to contain solution. This solution was mixed with 50 μL (100 pmol) of 2 pmol / μL fluorescent biotin solution, allowed to stand at room temperature for 20 minutes, and then measured for fluorescence intensity using Exfinite M200 (TECAN) at Ex = 460 nm and Em = 525 nm.

  The results are shown in FIG. As a result, the binding of TM2 with fluorescent biotin did not change at room temperature below 70 ° C. and retained 82% activity at 80 ° C. On the other hand, the binding activity between streptavidin and fluorescent biotin decreased by 40% at 70 ° C and 80% at 80 ° C. Further, the binding activity between avidin and fluorescent biotin decreased by 10% at 70 ° C. and 70% at 80 ° C. The temperature at which the reduction rate of the fluorescence intensity becomes 50% of the unheated sample was 85 ° C for TM2, whereas 73 ° C for streptavidin and 78 ° C for avidin.

1-5. Heat resistance of tamavidin 2 immobilized carrier
Preparation of tamavidin 2 magnetic beads 300 µl of magnetic beads coated with carboxyl groups (Dynabeads M-270 Carboxylic Acid, Dynal) were washed with 300 µl of 0.01N sodium hydroxide for 10 minutes, and then with 300 µl of ultrapure water for 10 minutes. Washed 3 times. 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (PIERCE) dissolved in cold ultrapure water was added to the washed magnetic beads to a final concentration of 0.2M. Shake for 30 minutes at room temperature. Thereafter, the magnetic beads were washed with 300 μl of cold ultrapure water and subsequently with 300 μl of 50 mM MES buffer (pH 5.0), and 300 μl (180 μg) of 0.6 mg / ml TM2 replaced with 50 mM MES buffer (pH 5.0). Mixed. By shaking at room temperature for 30 minutes, TM2 and magnetic beads were bound by covalent bond. The magnetic beads were collected with a magnet and the supernatant was removed. Next, the unreacted carboxyl groups of the beads were erased with 300 μl of 50 mM Tris buffer (pH 7.0), and then the magnetic beads were blocked with 300 μl of PBS buffer containing 0.5% BSA and 0.1% Tween20. The magnetic beads were suspended with 300 μl of PBS buffer to complete the magnetic beads. Magnetic beads were similarly prepared for streptavidin and avidin.

Heat test of tamavidin 2 magnetic beads (i) Heat resistance Using fluorescent biotin, the heat resistance of TM2 magnetic beads was measured using the streptavidin magnetic beads and avidin magnetic beads prepared above and commercially available streptavidin magnetic beads (Dynabeads M -270 Streptavidin, Dynal).

  Each magnetic bead was washed with PBS buffer, and then heated at room temperature, 70, 75, 80, 85, and 90 ° C. for 20 minutes. A solution containing each heated magnetic bead stepwise from 0 μL to 16 μL in 150 μL assay buffer was prepared. To this solution, 50 μL (50 pmol) of 1 pmol / μL biotin-4-fluorescein solution was mixed and allowed to stand at room temperature for 20 minutes, and then the fluorescence intensity of the supernatant was measured using Infinite M200 at Ex = 460 nm and Em = 525 nm.

  The results are shown in FIG. As a result, in the TM2 magnetic beads, the binding to fluorescent biotin was not changed from room temperature at 75 ° C. or lower, and the activity was 73% at 80 ° C. and 64% at 85 ° C. Streptavidin magnetic beads were inactivated by 70% at 80 ° C, and commercially available streptavidin magnetic beads (Dynal) were inactivated by 90% at 80 ° C. The avidin magnetic beads were deactivated 35% at 80 ° C. and 55% at 85 ° C. The temperature at which the fluorescence intensity decreases to 50% of the unheated sample is 87 ° C for TM2, whereas 78 ° C for streptavidin, 72 ° C for streptavidin (Dynal), and 84 ° C for avidin. there were.

(Ii) Biotin-binding activity in high temperature region Utilizing the property that fluorescence intensity of fluorescent biotin disappears when biotin binds to the biotin-binding site of biotin-binding protein, the biotin-binding ability of TM2 magnetic beads under high temperature conditions Were compared to commercially available streptavidin magnetic beads.

TM2 magnetic beads were prepared by covalently binding TM2 to magnetic beads (Dynabeads M-270 Carboxylic Acid, Dynal) whose surface was coated with carboxyl groups. As commercially available streptavidin magnetic beads, Dynabeads M-270 Streptavidin (Dynal) was used. TM2 magnetic beads are 2 × 10 ⁹ beads / 30 mg / mL, and 333 pmol of fluorescent biotin is bound per 1 mg of magnetic beads. Commercially available streptavidin magnetic beads are 6.7 × 10 ⁸ beads / 10 mg / mL, and 625 pmol of fluorescent biotin is bound per 1 mg of magnetic beads.

TM2 magnetic beads and 300 μL of commercially available streptavidin magnetic beads were washed twice with 300 μL of PBS buffer and suspended again in 300 μL of PBS buffer. Each magnetic bead was dispensed into 7 pieces of 42 μL. 1 μmol / μL fluorescent biotin solution (biotin-4-fluorescein: Molecular Probe) 50 μL (50 pmol) was added to assay buffer (50 mM NaH ₂ PO ₄ , 100 mM NaCl, 1 mM EDTA (pH 7.5)) 150, 149, 148, 146, 142, 140, and 134 μL, respectively, and preincubated at room temperature, 50, 60, 65, 70, 80, and 95 ° C. for 10 minutes. Subsequently, after preincubating both magnetic beads at room temperature, 50, 60, 65, 70, 80, and 95 ° C. for 5 minutes, 1, 2, 4, 8, 10, 16 μL was added to 50 pmol fluorescent biotin solution to obtain a final volume. 200 μL and they were kept heated at each temperature for 20 minutes. During this time, the mixture was suspended three times by pipetting. Thereafter, the magnetic beads were promptly collected with a magnet, and the fluorescence intensity of the supernatant was measured with Exfinite M200 (TECAN) at Ex = 460 nm and Em = 525 nm (Experiment 1). As Experiment 2, as in Experiment 1, room temperature, 70, 75, 80, 85, and 90 ° C. were analyzed.

  The results of Experiment 1 are shown in FIGS. 5A and B, and the results of Experiment 2 are shown in FIGS. 5C and D. As a result, in the TM2 magnetic beads, the binding with fluorescent biotin did not change at room temperature below 70 ° C., and retained 75% and 62% activities at 80 ° C. and 85 ° C., respectively (FIG. 5A). . On the other hand, in commercially available streptavidin magnetic beads, the binding with fluorescent biotin disappeared from 60% to 80% at 80 ° C. (FIG. 5B). The temperature at which the reduction rate of the fluorescence intensity becomes 50% of the unheated sample is 77.5 ° C. for streptavidin (Dynal), whereas that for tamavidin 2 is 87.5 ° C.

Preparation of TM2 Sepharose beads Separose beads whose surface carboxyl group was activated esterified with N-hydroxysuccinimide (NHS) (34 μm in diameter) HiTrap NHS-activated HP (GE Healthcare) was washed with 10 ml of cold 1 mM hydrochloric acid, Further, it was washed with 1 ml of cold ultrapure water. To this sepharose beads, 1 ml of 1 mg / ml TM2 previously dialyzed in 0.2 M sodium hydrogen carbonate buffer (pH 8.3) containing 0.5 M sodium chloride was mixed. TM2 and Sepharose beads were bound by shaking at room temperature for 3 hours. Next, unreacted active groups were erased with 5 ml of 50 mM Tris buffer (pH 8.0), and then sepharose beads were blocked with 5 ml of PBS buffer containing 0.5% BSA and 0.05% Tween 20. Sepharose beads were suspended in 1 ml of PBS buffer to complete TM2 Sepharose beads.

Heat test of TM2 Sepharose beads The heat resistance of TM2 Sepharose beads was verified using fluorescent biotin. TM2 Sepharose beads were washed with PBS buffer and then heated at room temperature, 70, 75, 80, 85, 90 ° C. for 20 minutes. A solution containing heated Sepharose beads stepwise from 0 μL to 16 μL in 150 μL assay buffer was prepared. Next, 50 μl (150 pmol) of 3 pmols / μl biotin-4-fluorescein solution was mixed and allowed to stand at room temperature for 20 minutes, and then the fluorescence intensity of the supernatant was measured using Infinite M200 at Ex = 460 nm and Em = 525 nm.

As a result, the binding with fluorescent biotin was not changed from room temperature at 80 ° C. or lower, and had 64% activity at 85 ° C. Further, the temperature at which the decrease rate of the fluorescence intensity becomes 50% of the unheated sample was 86 ° C., which showed the same heat resistance as the magnetic beads immobilized with TM2 (FIG. 6).

Example 2: Tamavidin 1 (TM1)
2-1. Characterization of tamavidin 1
E. coli expression and purification of tamavidin 1 When DNA encoding tamavidin 1 (SEQ ID NO: 1) is incorporated into the expression vector pTrc99A and expressed in E. coli, most of the expressed tamavidin 1 protein accumulates in the soluble fraction, and its expression The amount was high at the same level as tamavidin 2. Therefore, purification of recombinant tamavidin 1 was attempted as follows. After induction of expression, the collected E. coli pellet was suspended in 20 mM Kpi pH 7.0, and the cells were disrupted by ultrasound. The supernatant after centrifugation at 15000 rpm for 10 minutes was treated at 70 ° C. for 10 minutes. Centrifugation was further performed after the heat treatment, and the supernatant was replaced with 50 mM Tris-HCl pH 7.0, 50 mM NaCl. This sample was applied to an ion exchange column MonoQ HR5 / 5 (Phaemacia) equilibrated with the same buffer, and the recombinant tamavidin 1 was collected as a column passage fraction. The recovered amount of protein was approximately 1 mg per 50 mL of culture solution.

Mass spectrometry Mass spectrometry of tamavidin 1 was performed in the same manner as in Example 1. As a result, m / z = 15961.6 (corresponding to the monomer) and 31922.5 (corresponding to the dimer) were observed. The mass of the monomer was in good agreement with the molecular weight after tamavidin 1 was subjected to SDS-polyacrylamide electrophoresis. On the other hand, from the comparative analysis of the SDS-polyacrylamide electrophoresis image of the sample after adding an excess amount of biotin to tamavidin 1 and incubating it, and the electrophoresis image of tamavidin 2 subjected to the same treatment, tamavidin 1 is a tetramer. It was suggested that there is. Genetic information processing software GENETYX Ver. The isoelectric point of tamavidin 1 consisting of 143 amino acids in total was calculated to be 6.23 using 7 (Genetics).

Binding to biotin The fluorescence biotin binding activity of tamavidin 1 in the supernatant after centrifugation after the heat treatment at 70C was measured. The method was the same as that for tamavidin 2. Incubation with fluorescent biotin was performed at room temperature. The results are shown in FIG. It was revealed that tamavidin 1 binds to fluorescent biotin even after treatment at 70 ° C.

2-2. Heat resistance of tamavidin 1
Protein Stability Purified tamavidin 1 at a concentration of 0.2 μg / μL was heated at room temperature, 50, 60, 70, 80, 90, 99 ° C. for 20 minutes at 10 μL (2 μg). Subsequently, the mixture is centrifuged at 15000 rpm for 10 minutes, and the soluble protein in the supernatant is suspended with an equal amount of 2 × SDS sample buffer (100 mM Tris-HCl pH 6.8, 12% 2-mercaptoethanol, 2% SDS, 20% glycerol). After becoming cloudy and heating at 95 ° C. for 10 minutes, SDS-PAGE was performed. The protein band was detected by CBB staining. A calibration curve was created based on a quantitative marker (LMW ELECTROPHORESIS CALIBRATION KIT; Pharmacia Biotech) using Las-3000 (FUJIFILM), and the protein band was quantified. As a result, the temperature at which 50% protein of tamavidin 1 disappeared was 76 ° C. As a result, it has been clarified that tamavidin 1 has a heat resistance of 5 ° C. higher than that of the aforementioned streptavidin. Moreover, when biotin was added to tamavidin 1, the heat resistance became extremely strong, and the tetramer was not dissociated even after treatment at 95 ° C.

  Since tamavidin linked to the solid support of the present invention retains protein stability and biotin-binding property at high temperatures, the solid support of the present invention can be used in assay systems including treatment at high temperatures. Assay systems involving high temperature processing contribute to the speeding up of a series of protocols to allow separation, concentration, purification, detection and / or capture of highly specific substances. In addition, tamavidin has higher production efficiency than egg white-derived avidin or streptavidin, so the solid support of the present invention is advantageous in terms of cost compared to a solid support linked with a commercially available biotin-binding protein. .

Claims (9)

A solid support, the following:
a) a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4;
b) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4;
c) a protein comprising an amino acid sequence having at least 60% sequence identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; and d) a stringent strand complementary to the base sequence of SEQ ID NO: 1 or 3 A protein comprising an amino acid sequence encoded by a nucleic acid that hybridizes under mild conditions;
The solid support, wherein a thermostable biotin-binding protein selected from the group consisting of is linked.   The solid support according to claim 1, which retains biotin-binding ability even after heat treatment at 70 ° C to 90 ° C.   The solid support according to claim 1, which has a biotin-binding ability even under heating conditions of 70 ° C to 90 ° C. Solid carrier, cellulose, Teflon ^TM, nitrocellulose, agarose, dextran, chitosan, polystyrene, polyacrylamide, polyester, polycarbonate, polyamide, polypropylene, nylon, polydiene vinylidene difluoride, latex, silica, glass, glass fiber, gold, A material selected from the group consisting of platinum, silver, copper, iron, stainless steel, ferrite, silicon wafer, polyethylene, polyethyleneimine, polylactic acid, resin, polysaccharide, protein (eg albumin), carbon and combinations thereof. The solid support according to any one of claims 1 to 3, which is mainly constituted.   The solid support according to any one of claims 1 to 3, wherein the solid support is selected from the group consisting of beads, magnetic beads, thin films, microtubules, filters, plates, microplates, carbon nanotubes, and sensor chips.   Use of the solid support according to any one of claims 1 to 5, characterized in that it is exposed to heating conditions of 70 ° C to 90 ° C. A method for separating, concentrating, capturing, purifying and / or detecting a substance linked to biotin, comprising the following steps:
1) The solid support according to any one of claims 1 to 6 is contacted with a substance linked to biotin, and the substance linked to biotin is bound to the solid support;
2) washing away contaminants not bound to the solid support; and 3) separating, concentrating, capturing or purifying the material by recovering the material linked to biotin bound to the solid support, and / or Detect the substance;
Wherein at least one of the steps is performed under heating conditions of 70 ° C to 90 ° C.   The method of Claim 7 whose heating conditions are 75 to 85 degreeC.   The method according to claim 7, wherein the substance linked to biotin is a nucleic acid linked to biotin.

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