JP2002187899A - Peptide having binding ability to luciferase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peptide having binding ability to luciferase and capable of being used for directly quantitating the luciferase. SOLUTION: This peptide having binding ability to the luciferase is composed of a peptide chain comprising the whole sequence or a partial sequence of at least one kind of an amino acid sequence selected from 8 kinds of amino acid sequences such as an amino acid sequence represented by the following: Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Leu-Ala-Arg-Trp and the luciferase-binding peptide enables to detect the luciferase with high quantitative accuracy by using a method such as a competition method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to a peptide containing an amino sequence capable of binding to luciferase, and a luciferase detection kit using the peptide having binding ability to luciferase as a detection means.

[0002]

2. Description of the Related Art Luciferase is a general term for oxygenase that catalyzes bioluminescence, and is also called a luminescent enzyme. This enzyme catalyzes the oxidation of luciferin by oxygen molecules, and with the chemical change at that time, chemiluminescence derived from the substrate luciferin is obtained. The reaction mechanism is
It is known that luciferase varies depending on the species from which it originated. Among them, the bioluminescence reaction by luciferase isolated from fireflies has been most studied. Firefly luciferase (EC.1.13.12.7) acts on luciferase substrates typified by adenosine triphosphate (ATP) and firefly luciferin to generate adenosine monophosphate (AMP) and oxyluciferin and at the same time emit light.

[0003] The bioluminescence reaction using luciferase is very useful as an analytical method because it is more advantageous in terms of sensitivity than chemiluminescence using peroxidase. The most typical field of use of luciferase is as an analytical reagent. First, specific examples of the analysis reagent include various binding analysis systems using luciferase as a labeling substance. The binding analysis system includes an immunological assay using an antigen-antibody reaction, a nucleic acid hybridization assay using the affinity of a complementary nucleic acid, and avidin-biotin, a hormone-hormone receptor, and a sugar-lectin. Analytical systems and the like utilizing binding between substances having such specific affinity are known. If luciferase is used as a labeling substance (labeling enzyme) in these binding assay systems, the enzyme activity is measured to determine the amount of finally bound (or unbound) substance in the binding assay system. Can be tracked.

[0004] In addition to the use of the bioluminescence reaction by luciferase, use as a means for detecting luciferase itself or ATP used in the enzymatic reaction can be considered. For example, when screening for a foreign gene using the luciferase gene as an index or analyzing a gene mutation, it is necessary to track the activity of the luciferase produced.

[0005] In addition, A using firefly luciferase
The method of detecting TP is used for measuring the number of cells, tracking an enzyme reaction using ATP as an enzyme reaction product as an index, and a binding analysis system using ATP itself as a label. More specifically, by measuring the concentration of ATP contained in the system, an indirect method for measuring the number of microorganisms contained in water, food, cosmetics, etc., a method for measuring the activity of cell tissues such as blood cells and sperm, drugs It has been reported that the method can be applied to the measurement of the degree of influence of stress.

[0006] On the other hand, luciferase itself is generally determined based on its function and enzyme activity. Japanese Patent Application Laid-Open No. 07-069899 discloses that the common logarithm of the luciferase concentration and the common logarithm of the enzyme activity (integrated fluorescence value) of the enzyme are linearly plotted as a linear function. It is disclosed that it is possible. For example, for luciferase expressed in infected cells, the activity value (integrated fluorescence value) is measured, and the amount of protein is determined based on the above-mentioned calibration curve.

In Japanese Patent Application Laid-Open No. 08-116991, the amount of luciferase contained in the transgenic nematode body is determined by utilizing the fact that the amount of luminescence depends on the amount of luciferase added under certain conditions. It is measured as an increase in the amount of luminescence in the cell extract.

[0008]

The method for quantifying luciferase based on its enzyme activity (emission amount) presupposes that the conditions for preparing a calibration curve and the conditions for sample measurement are the same. However, it is often difficult to guarantee this prerequisite when quantifying the amount of luciferase in vivo, for example in a recombinant. In addition, luciferase extracted from biological materials such as fireflies has a problem with its stability and its enzyme activity decreases with time. There is naturally a limit to the reliability of

Therefore, instead of the method of quantification based on the enzyme activity (the amount of luminescence), a method of quantifying luciferase, more specifically, a method of detecting luciferase using a peptide having a binding property to luciferase as a detection means. A proposal for a quantitative method is desired.

An object of the present invention is to provide a peptide capable of binding to luciferase, which can be used for direct quantification of luciferase, and to provide a peptide capable of binding to luciferase. An object of the present invention is to provide a method for detecting luciferase using a peptide having the following as a detection means, and a detection kit exclusively used for the method. The peptide having a binding property to luciferase, which is an object of the present invention, is a useful means not only for directly quantifying luciferase in a sample, but also for separating and purifying luciferase.

[0011]

Means for Solving the Problems In order to solve the above problems, the present inventors proceeded with screening of peptides having specific binding to luciferase from a random peptide library, and found that a total of 8 Some peptides were found to have sufficiently high binding capacity. The amino acid sequences of the eight peptides were analyzed, and the peptide chains considered to be involved in the binding to luciferase were identified. Specifically, the peptide chains (I) to (VIII) shown below were found to have high luciferase binding properties. Peptide (I): Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Le
u-Ala-Arg-Trp peptide (II): Trp-His-Asn-Trp-Thr-Trp-Thr-Gly-Le
u-Pro-Gly-Gln peptide (III): Gly-His-Trp-Thr-Ala-Ser-Thr-Asn-T
yr-Val-Arg-Pro peptide (IV): Gln-Thr-Thr-His-Trp-Asp-Ser-Ala-Ly
s-Tyr-Pro-Ala peptide (V): Phe-His-Glu-Asn-Trp-Pro-Ser peptide (VI): Phe-His-Trp-Trp-Tyr-Thr-Ile peptide (VII): Phe- Pro-Ala-His-Ala-Ser-Arg peptide (VIII): Tyr-Ser-Phe-Glu-Arg-Ser-Lys When the analyzed amino acid sequences are compared with each other, peptides (I) to (VIII) Have no significant homology between them, and it was judged that the specific binding to luciferase was achieved by the partial sequence contained in each amino acid sequence. The present inventors have completed the present invention based on such findings. Each has a different amino acid sequence.

That is, the peptide having a binding property to the luciferase of the present invention comprises all or at least one amino acid sequence selected from the group consisting of the following eight amino acid sequences (I) to (VIII): It is a peptide having a binding property to luciferase, comprising a peptide chain comprising a partial sequence having an amino acid length of 5 or more. Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Leu-Ala-Arg-Trp (I) Trp-His-Asn-Trp-Thr-Trp-Thr-Gly-Leu-Pro-Gly-Gln ( II) Gly-His-Trp-Thr-Ala-Ser-Thr-Asn-Tyr-Val-Arg-Pro (III) Gln-Thr-Thr-His-Trp-Asp-Ser-Ala-Lys-Tyr-Pro- Ala (IV) Phe-His-Glu-Asn-Trp-Pro-Ser (V) Phe-His-Trp-Trp-Tyr-Thr-Ile (VI) Phe-Pro-Ala-His-Ala-Ser-Arg ( VII) Tyr-Ser-Phe-Glu-Arg-Ser-Lys (VIII)

For example, the peptides having a binding property to the luciferase of the present invention have the following amino acid sequences (I): Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Leu-Ala-Arg -A peptide comprising a peptide chain comprising a partial sequence of Trp (I) or its partial sequence having an amino acid length of 5 or more, the following amino acid sequence:
(II): a peptide comprising a peptide chain comprising the whole of Trp-His-Asn-Trp-Thr-Trp-Thr-Gly-Leu-Pro-Gly-Gln (II) or a partial sequence thereof having an amino acid length of 5 or more. , The following amino acid sequence
(III): a peptide comprising a peptide chain comprising the whole of Gly-His-Trp-Thr-Ala-Ser-Thr-Asn-Tyr-Val-Arg-Pro (III) or a partial sequence thereof having an amino acid length of 5 or more. , The following amino acid sequence
(IV): a peptide comprising a peptide chain comprising the whole of Gln-Thr-Thr-His-Trp-Asp-Ser-Ala-Lys-Tyr-Pro-Ala (IV) or a partial sequence thereof having an amino acid length of 5 or more. , The following amino acid sequence
(V): Phe-His-Glu-Asn-Trp-Pro-Ser (V) or a peptide comprising a peptide chain comprising a partial sequence having an amino acid length of 5 or more, the following amino acid sequence:
(VI): Phe-His-Trp-Trp-Tyr-Thr-Ile (VI) or a peptide consisting of a peptide chain comprising a partial sequence having an amino acid length of 5 or more, the following amino acid sequence:
(VII): Phe-Pro-Ala-His-Ala-Ser-Arg (VII) or a peptide consisting of a peptide chain comprising a partial sequence having an amino acid length of 5 or more, or the following amino acid sequence (VIII) The peptide can be a peptide consisting of a peptide chain comprising the entirety of Tyr-Ser-Phe-Glu-Arg-Ser-Lys (VIII) or a partial sequence thereof having an amino acid length of 5 or more.

[0014] In addition, the present invention also provides a method for using the above-mentioned peptide having a binding property to luciferase for the detection of luciferase, and also provides a kit used for this detection method. That is, the luciferase detection kit of the present invention provides, as a luciferase detecting means, all or at least one amino acid sequence of at least one amino acid sequence selected from the group consisting of the following eight amino acid sequences (I) to (VIII): A luciferase detection kit comprising a peptide having a binding property to luciferase, comprising a peptide chain comprising the above partial sequence. Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Leu-Ala-Arg-Trp (I) Trp-His-Asn-Trp-Thr-Trp-Thr-Gly-Leu-Pro-Gly-Gln ( II) Gly-His-Trp-Thr-Ala-Ser-Thr-Asn-Tyr-Val-Arg-Pro (III) Gln-Thr-Thr-His-Trp-Asp-Ser-Ala-Lys-Tyr-Pro- Ala (IV) Phe-His-Glu-Asn-Trp-Pro-Ser (V) Phe-His-Trp-Trp-Tyr-Thr-Ile (VI) Phe-Pro-Ala-His-Ala-Ser-Arg ( VII) Tyr-Ser-Phe-Glu-Arg-Ser-Lys (VIII)

[0015] In order to name the amino acid residues that participate in the construction of the peptide according to the present invention, NUCLEIC ACIDS RESERCH 13, 3021
-3030 (1985) and THE BIOLOGICAL JOURNAL 219, No. 2, 345-371 (19)
The IUPAC-IUB Rules (Rules) described in 84) are used.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a peptide having a binding property to luciferase of the present invention, a method for preparing the same, and a method for using a luciferase-binding peptide in a luciferase detection kit of the present invention will be described in more detail. .

The peptide having a binding property to luciferase of the present invention is a peptide chain comprising at least one of the above eight amino acid sequences (I) to (VIII) or a partial sequence having an amino acid length of 5 or more. It is composed of That is, it may be present as a peptide containing at least a part of the peptide chain constituting the amino acid sequence exhibiting the binding property to luciferase. When such a peptide chain containing an amino acid sequence exhibiting the binding to luciferase as a partial sequence thereof, the remaining amino acid sequence may be any amino acid sequence, as long as the binding to luciferase is not inhibited. Is also good.

The luciferase-binding peptide according to the invention can be prepared according to its entire amino acid sequence by any of the known organic chemical peptide synthesis methods or by recombinant DNA.
It can be prepared using techniques.

The method of organic peptide synthesis involves combining the required amino acids by a condensation reaction in a homogeneous phase or using a so-called solid phase.

The condensation reaction used for elongating a peptide chain includes the steps of: a) converting a compound (amino acid or peptide) having a free carboxyl group and other reactive groups having a protected amino group to a free amino group; The other reactive group is condensed with the protected compound (amino acid or peptide) in the presence of a condensing agent, or b) has an activated carboxyl group and is the other reactive group free? Or condensing a protected compound (amino acid, peptide) with a free or protected compound (amino acid, peptide) having a free amino group and other reactive groups ) Or b).

In the above-mentioned method b), the activation of the carboxyl group may be carried out by, in particular, converting the carboxyl group to an acid halide, azide, acid anhydride, imidazolide, or N-hydroxy-succinimide, N-hydroxy-benzotriazole or p-type. -By conversion to an activated ester such as nitrophenyl ester.

Among these, the most common methods for causing the above condensation reaction are the carbodiimide method, the azide method, the mixed acid anhydride method, and the E.C. Gross and J.M. Meie
nhofer, The Peptides, Ana
lysis, Synthesis, Biolog
y, vols. 1-3, Academic Pres
s Inc. , 1979, 1980, 1981, using an activated ester.

In preparing suitable fragments of the above-described peptides according to the present invention using the "solid phase method", peptide synthesis methods that can be used are described, for example, in J. Am. Amer. Che
m. Soc. 85, p. 2149, 1963 and I
nt. J. Peptide Protein Res.
35, pp. 161-214, 1990. In this “solid phase method”, the amino acid binding (elongation of the peptide chain) of the peptide to be prepared is usually
Starting from the carboxyl terminal side. In this case, the synthesis method by the “solid phase method” requires a solid phase that carries a reactive group or onto which a reactive group can be introduced.
For the solid phase, for example, a copolymer of benzene and divinylbenzene with reactive chloromethyl groups or a polymer solid phase imparted with the property of reacting with hydroxymethyl or amine functions can be used. Particularly suitable solid phases are described, for example, in J. Am. Am. Chem. Soc. 95, p.
P-alkoxybenzyl alcohol resin (4-hydroxy-methyl-phenoxy-methyl-copolystyrene-1% divinylbenzene resin) described by Wang in 1328, 1974.

After a series of synthesis (elongation of the peptide chain), the peptide can be separated from the solid phase under mild conditions. Specifically, after the synthesis of the desired amino acid sequence, the peptide is subsequently separated from the resin in the solid phase with, for example, trifluoromethanesulfonic acid or methanesulfonic acid dissolved in trifluoroacetic acid. Alternatively, the peptide can be separated from the support by a transesterification reaction with a lower alcohol, preferably methanol or ethanol, in which case the separated peptide will be in the form of a lower alkyl ester. Similarly, the peptide can be separated using ammonia, in which case the separated peptide has an amide form at the C-terminus.

As described above, a reactive group which does not participate in the above condensation reaction (elongation of the peptide chain), specifically, a reactive group on an amino acid residue and an amino group at the N-terminal of the peptide chain, It is effectively protected by a group which can be very easily removed again by hydrolysis or reduction with acids, bases. For example, a carboxyl group can be effectively protected by esterification with, for example, methanol, ethanol, t-butanol, benzyl alcohol or p-nitrobenzyl alcohol, and an amine attached to a solid support. On the other hand, a group that can effectively protect the amino group is
Ethoxycarbonyl, benzyloxycarbonyl, t-
A butoxy-carbonyl (t-boc) or p-methoxy-benzyloxycarbonyl group or an acid group derived from a sulfonic acid such as a benzene-sulfonyl or p-toluene-sulfonyl group, but substituted or unsubstituted. It is also possible to use other groups such as aryl or aralkyl groups such as benzyl and triphenylmethyl, and groups such as ortho-nitrophenyl-sulphenyl and 2-benzoyl-1-methyl-vinyl.
In particular, suitable α-amino protecting groups include, for example, base-sensitive 9-fluorenyl-methoxycarbonyl (Fmoc)
Group (Carpino and Han, J. Amer. C
hem. Soc. 92, p. 5748, 1970)
It is.

A more extensive description of protecting groups that can be used in peptide synthesis is given in Gross, Ude
nfriend and Meienhofer eds., The
Peptides, Analysis, Synth
ess, Biology, vols. 1-9, Ac
ademic Press Inc. , 1979-19
87.

For example, it is necessary to protect the ε-amino group of lysine in addition to the α-amino group, and it is appropriate to protect the guanidine group of arginine. Protecting groups commonly used for such protection include Boc group for lysine, Pmc group, Pms group for arginine and M
a bs group or an Mtr group.

These protecting groups may be prepared in a variety of conventional ways depending on the nature of the group, for example with trifluoroacetic acid or by mild reduction with hydrogen and a catalyst such as palladium, or in glacial acetic acid. HBr can be removed.

The luciferase-binding peptide according to the present invention can be combined into a plurality of amino acid sequences having luciferase-binding properties so as to form a single molecule as a series of amino acid sequences. The two or more partial peptide chains correspond to those covalently linked, to be a hybrid peptide or a complex peptide, for example, using the method described above, according to a series of amino acid sequence of interest, This is possible by solid phase peptide synthesis. At this time, the amino acid sequences corresponding to the individual partial peptide chains are aligned with each other. In addition,
Between amino acid sequences corresponding to individual partial peptide chains,
A linker sequence can also be inserted. Such a linker sequence may utilize, for example, a stretch of 2-5 residues of glycine.

Furthermore, hybrid or composite peptides can also be prepared by solid phase synthesis using fragment condensation techniques. In advance, the amino acid sequence is (I)-(VIII)
A plurality of fragments each having a predetermined amino acid sequence, including a fragment (partial peptide chain) containing any of the above amino acid sequences, is individually prepared and purified. Thereafter, the fragments (partial peptide chains) are condensed to form a whole peptide having a desired series of amino acid sequences. This fragment condensation technique is preferred when relatively long hybrid or complex peptide sequences are synthesized. Methods for preparing relatively long peptides are known to those skilled in the art and are described, for example, in The Peptides, A.
analysis, Synthesis, Biolog
y, vols. 1-9 (see above).

Alternatively, in addition to the above-described series of long peptide chains, the luciferase-binding peptide of the present invention may be prepared as a hybrid or complex peptide by bonding appropriately modified peptide chains of the present invention. it can.

For example, if the peptide chain to be linked does not itself contain a cysteine amino acid in its amino acid sequence, one of the preferred ways to link the two different peptide chains is to link each peptide chain to This is a method of converting into a derivative having an additional cysteine residue at the carboxyl or amino terminus and forming a disulfide bond between the additional cysteine residues. In this method for forming a disulfide bond, after conversion into a derivative having a cysteine residue at the terminal, the thiol functional group of the introduced single cysteine at the terminal is converted to 2,2'-dithiol to one peptide derivative. Activate with dipyridine. The resulting pyridyl-dithio-peptide derivative is
Reaction with a second peptide derivative having a cysteine thiol group gives a hybrid peptide in which the individual peptide chains are linked by disulfide bonds.

[0033] Various other methods for preparing hybrid peptides are also available. For example, chemical methods developed in the field of protein-protein binding can be used. Regarding such a technique of linking between peptide chains, M
eans and Feeney (Bioconj. Che)
m. 1 pp. 2-12, 1990). For example, if a well-known homo- or heterobifunctional cross-linking agent is used, individual peptide chains can be linked by a disulfide bond, a thioether bond, or an amide bond.

After the synthesis, all the protecting groups are removed and the crude product released from the solid support is once lyophilized. The crude product is then purified by medium pressure liquid chromatography to obtain a peptide of about 70% purity. The crude product is then purified by high pressure liquid chromatography (HPLC) or by another type of chromatography to yield a peptide of 95% purity in a purity assay by analytical HPLC.

As mentioned above, the luciferase binding peptides according to the invention can also be prepared using recombinant DNA technology. The availability of this recombinant DNA technology is when a partial peptide chain of the amino acid sequence of interest is incorporated "in tandem" into a repetitive sequence, or when a partial peptide chain is incorporated into a protein or polypeptide having a much larger overall amino acid length. When it can be prepared as a component, or as a fusion protein with a general-purpose fusion partner, for example (part of) β-galactosidase, or can be replaced with an amino acid sequence of a hypervariable region in another type of antibody to obtain a recombinant type. This is particularly important when creating an artificial antibody. Therefore, as described above, the main part is derived from other proteins and polypeptides, and a peptide in which a partial peptide chain of the target amino acid sequence is inserted and added as a part thereof is also within the technical scope of the present invention. included. In this preparation method, a luciferase-binding peptide according to the present invention is characterized as a component of the recombinant DNA.
A nucleic acid sequence encoding any of the above eight amino acid sequences (I) to (VIII) or a partial peptide chain containing a partial sequence having an amino acid length of 5 or more is used.

The method using the above recombinant DNA technology is as follows.
Preparing the desired peptide by expressing a recombinant polynucleotide containing a nucleic acid sequence encoding one or more luciferase-binding peptide chain portions to be prepared in a microorganism suitable as a host.

The nucleic acid sequence encoding the luciferase-binding peptide chain portion according to the present invention is inserted into a commonly used plasmid or the like as a DNA fragment having the nucleotide sequence, and various replication realization D provided in such a plasmid or the like is performed.
By linking with an NA sequence, a so-called recombinant vector molecule can be obtained. This recombinant vector molecule can be used for transformation of a suitable host. Useful recombinant vector molecules are preferably derived, for example, from plasmids, bacteriophages, cosmids or viruses.

Specific vectors or cloning vehicles that can be used for cloning the nucleic acid sequence of interest are known to those of skill in the art and include, among others, pBR322, various pUCs,
Plasmid vectors such as pGEM and Bluescript plasmids, bacteriophages such as k
ph-derived phages derived from gt-Wes, Charon 28 and M13, as well as viral vectors such as SV40, adenovirus or polyomavirus (RL Rodriquez and DT Den).
Hardt ed., Vectors: A survey o
f molecular cloning vector
s and their uses, Butterworth
ths, 1988; A. Lenstra et al.
Arch. Virol. 110, pp. 1-24,
1990). Techniques to be used for the construction of recombinant vector molecules are known to those skilled in the art,
Maniatis et al. (Molecule
ar Cloning: A Laboratory Ma
natural, second edition, Cold
Spring Harbor Laboratory,
1989).

For example, a luciferase-binding peptide chain portion according to the present invention may be a nucleic acid sequence (D
Inserting an NA fragment) into a cloning vector involves cutting both ends of the gene (DNA) to be inserted with one or more restriction enzymes, if necessary, and cloning the desired cloning vehicle ( site)
Are also digested with the same restriction enzyme to form complementary DNA ends at the ends where they are to be ligated to each other, so that insertion and ligation can be easily performed.

The recombinant vector molecule is pBR3
One or more marker activities (genes) that can be used to select the desired transformants, such as 22 ampicillin and tetracycline resistance, and the ampicillin resistance of pUC8 and the activity of the α-peptide coding region of β-galactosidase. It may additionally have.

The luciferase-binding peptide according to the present invention may be provided on the peptide chain in vivo or in vitro by glycosylation, amidation, carboxylation or phosphorylation, as long as the amino acid sequence conferring luciferase binding is maintained. It is also possible to modify with. Therefore, the luciferase-binding peptide according to the present invention includes, as a part of the present invention, a functionally modified product such as an acid addition salt, an amide, an ester, particularly a C-terminal ester, and an N-acyl derivative. .

In the luciferase-binding peptide according to the present invention, for example, as described above, when a partial peptide chain having an objective amino acid sequence is contained as a component of a natural protein or polypeptide, the entire peptide chain It is understood that some natural modifications may be present. Such alterations can be manifested by one or more amino acid differences (substitutions), or by the deletion or insertion of one or more amino acids, in the entire amino acid sequence. Heretofore, various types of amino acid substitutions which do not substantially alter the biological activity and immunological activity of natural proteins and polypeptides have been disclosed. More specifically, typical amino acid substitutions between related amino acids, or substitutions that often occur during evolution, include, for example, Ser / Al
a, Ser / Gly, Asp / Gly, Asp / As
n, Ile / Val (MD Dayhof,
“Atlas of protein sequence
and structure, "Nat. Biome
d. Res. Found. , Vol. 5, supp
l. 3, Washington D. C. , 1978
reference). For example, based on this information, Lipman and Pearson developed a rapid and sensitive protein comparison method (Science 227, pp. 1434).
-1441, 1985), and the functional similarity between homologous proteins is measured. The present invention can also include mutations found at a high frequency in the natural world as long as the luciferase binding property is not impaired.

The luciferase-binding peptide of the present invention has a specific binding property to luciferase in spite of a relatively short peptide chain, and thus can be easily prepared, and can be used as a probe, for example, on a substrate. It has the advantage that it is easy to fix to If the luciferase-binding peptide is supported on a support and brought into contact with a sample containing luciferase, the luciferase contained in the sample can be captured in the vicinity of the support via the bond with the luciferase-binding peptide. it can. in this way,
Although the upper limit of the amino acid length of the luciferase-binding peptide of the present invention is not particularly limited, when it is used as an immobilized probe, the amino acid length is preferably 50 or less, more preferably 30 or less. . That is, when a peptide having a shorter chain length which does not exceed the amino acid length is used, the binding peptide can be supported on the surface of the support at a high density, so that luciferase can be captured at a high density near the support. Can be. Therefore, if a competitive reaction with a known concentration of luciferase having a labeling substance, or a sandwich reaction or agglutination reaction with a free peptide (labeling probe) having a labeling substance is used, in vitro in a biological sample can be performed. It can be used for highly sensitive detection and quantification of luciferase.

In carrying the luciferase-binding peptide of the present invention, supports that can be used include, for example, the inner walls, tubes or capillaries of micro test wells or cuvettes,
Membranes, filters, test strips, and even particles such as latex particles, aldehyde particles;
Surfaces such as ceramic magnetizable particles having active aldehyde surface groups, dye sols, metal compounds as metal sols or sol particles, and carrier proteins such as bovine serum albumin. On the other hand, the labeling substance that can be used in the above detection / quantification method is, inter alia, a fluorescent compound, a radioisotope, a labeling enzyme, a dye sol, a metal sol, or a metal compound as a sol particle.

The binding reaction between the luciferase-binding peptide of the present invention and luciferase is carried out in a suitable buffer at pH 5 to 9, preferably pH 6 to 7.5, at a temperature of 2 to 40.
C., preferably 4 to 10.degree. C., usually for 10 minutes to 24 hours, preferably 30 minutes to 60 minutes. The buffer used is not particularly limited, but is widely used in the above-mentioned pH range. For example, a phosphate buffer or a Tris-HCl buffer having a concentration of about 0.01 to 1 M, preferably about 0.1 to 0.5 M is suitable. is there.

After the reaction for forming a conjugate with luciferase and the luciferase-binding peptide of the present invention supported on a support, and / or after the reaction between the formed conjugate and the labeled probe, the reaction product Is washed with an appropriate washing solution, for example, distilled water, physiological saline, a phosphate buffer, a buffer containing Triton X-100 or a buffer containing Tween 20, to remove unreacted substances. The remaining binding complex is washed, after washing, depending on the label, a measuring instrument suitable for evaluating the enzyme activity, radioactivity, or fluorescence of the labeling enzyme, for example, a spectrophotometer for measuring the enzyme reaction product, a well The presence or amount of the labeling substance attached to the binding complex is measured using a type scintillation counter, a fluorometer, or the like.

The labeled probe used for the sandwich reaction with the free peptide (labeled probe) provided with the above-mentioned labeling substance includes a peptide which selectively binds to the luciferase constituting the binding complex, ie, The luciferase-binding peptide of the present invention is used. At that time, a combination of a luciferase-binding peptide supported on a support used for forming a binding complex and a luciferase-binding peptide used for a labeled probe is selected so that the binding sites do not compete with each other. I do.
Therefore, the two types of luciferase-binding peptides are selected from those having different amino acid sequences involved in the luciferase-binding properties.

For example, a luciferase-binding peptide supported on a support may include, in addition to the amino acid sequence portion involved in the luciferase binding property,
Alternatively, it may include additional peptide moieties utilized for binding and immobilization. For example, by adding cysteine to the N-terminus or C-terminus, it is possible to autonomously form a peptide monolayer by utilizing the ability of the SH group of the added cysteine side chain to bind to the gold surface. . On the other hand, the luciferase-binding peptide used for the labeling probe can also include, if necessary, an additional peptide portion used for attaching a labeling substance, in addition to the amino acid sequence portion involved in luciferase binding. .

According to the luciferase detection kit of the present invention, a luciferase-binding peptide carried on a support and a standard luciferase provided with a labeling substance, or a free luciferase provided with a labeling substance, can be used in accordance with the detection method exemplified above. A kit comprising a luciferase-binding peptide (labeled probe) can be provided.

[0050]

The present invention will be described more specifically below with reference to examples. Although these examples are examples of the best mode of the present invention, the present invention is not limited to these specific examples.

Example 1 The above amino acid sequences (I) to (VII)
Eight kinds of peptides each having I) were chemically synthesized, and their binding to luciferase was verified.

Synthesis of Peptide The peptide was chemically synthesized by a method in which amino acids were bonded one by one to the amino acid derivative fixed to the resin from the carboxyl terminal side (solid phase synthesis method). As an amino acid used in each cycle, a special α-amino acid derivative in which an α-amino group and a reactive group in an amino acid residue portion (side chain) were blocked with a protecting group was used. In this example, a derivative of each amino acid in which each α-amino group was blocked by an Fmoc group (9-fluorenylmethyloxycarbonyl group) was used (Fmoc method). Peptide synthesis (elongation of the peptide chain) is based on the α-amino group of the amino acid bound to the resin.
The reaction of deprotecting the Fmoc group and then coupling the amino acid derivative having the activated carboxyl group to the deprotected α-amino group was sequentially repeated. Each peptide was synthesized by the above-mentioned Fmoc solid phase synthesis method using a peptide synthesizer (PSSM-8: manufactured by Shimadzu Corporation).

That is, in the synthesis of the peptide (I) having the following amino acid sequence, Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Leu-Ala-Arg-Trp (I) Amino acid corresponding to the C-terminal residue (Tr
Fmoc-Trp-resin resin into which p) has been introduced (0.44 m
mol / g; 30 mg of Shimadzu Corporation) was set in the reaction vessel of the peptide synthesizer, and washed once with dimethylformamide (hereinafter referred to as "DMF"). Next, a deprotection solution (30% (v / v) piperidine / DMF) was reacted twice for 5 minutes and 3 minutes to remove the Fmoc group of the amino acid (Trp) bound to the resin.
And washed 5 times. Second amino acid from the C-terminal side (Arg)
150 μmol of Fmoc-Arg (Pmc) -OH / PyBOP corresponding to
Activated by adding an activator solution (0.5 M 1-hydroxybenzotriazole (HOBt) / DMF and 1 M N-methylmorpholine / DMF) to
Amino acids bound to the resin excluding the Fmoc group (Tr
p) was reacted for 30 minutes at room temperature. Here, unreacted Fmo
In order to remove a reaction solution containing c-Arg (Pmc) -OH / PyBOP and the like, the generated Fmoc-Arg (Pmc) -Trp-resin resin was washed four times with DMF. Then, the Fmoc-Arg (Pmc) -Trp-resin resin was again subjected to deprotection of its N-terminal Fmoc group under the above-mentioned conditions, washed with DMF five times, and then activated with an activator solution. Reaction with -OH / PyBOP solution. After the completion of the reaction, the resultant was washed four times with DMF to remove a reaction solution containing unreacted Fmoc-Ala-OH / PyBOP and the like.

In the subsequent elongation of the peptide chain, the same operation is repeated to obtain the desired protected peptide resin: Fm
oc-Lys (Boc) -His (Trt) -Val-Asn (Trt) -Glu (OtBu) -Leu-Ar
g (Pmc) -Glu (OtBu) -Leu-Ala-Arg (Pmc) -Trp-resin resin was synthesized.

The amino acid derivatives used in the peptide synthesis in the following Examples are as follows (the parentheses attached to each amino acid indicate a protecting group for protecting the reactive group of the amino acid residue portion (side chain)). Fmoc-Ala-OH / PyBOP, Fmoc-Arg (Pmc) -OH / PyBOP, Fmoc-Asn (Trt) -OH / PyBOP, Fmoc-Asp (OtBu) -OH / PyBOP, Fmoc-Cys (Trt) -OH / PyBOP, Fmoc-Gln (Trt) -OH / PyBOP, Fmoc-Glu (OtBu) -OH / PyBOP, Fmoc-Gly-OH / PyBOP, Fmoc-His (Trt) -OH / PyBOP, Fmoc-Ile-OH / PyBOP, Fmoc-Leu-OH / PyBOP, Fmoc-Lys (Boc) -OH / PyBOP, Fmoc-Met-OH / PyBOP, Fmoc-Phe-OH / PyBOP, Fmoc- Pro-OH / PyBOP, Fmoc-Ser (tBu) -OH / PyBOP, Fmoc-Thr (tBu) -OH / PyBOP, Fmoc-Trp-OH / PyBOP, Fmoc-Tyr (tBu) -OH / PyBOP, Fmoc-Val -OH / PyBOP.

The meanings of the abbreviations used in the above description are as follows: Trt = trityl; Pmc = 2,2,5,7,8-pentamethylchroman-6
Boc = t-butoxycarbonyl; tBu = tert-butyl; PyBOP = benzotriazol-1-yl-oxy-tris (pyrrolidino) phosphonium hexafluorophosphate; OtBu = tert-butoxy.

After a series of elongation of the peptide chain is completed, the synthesized protected peptide resin: Fmoc-Lys (Boc) -His (T
(rt) -Val-Asn (Trt) -Glu (OtBu) -Leu-Arg (Pmc) -Glu (OtBu)-
The deprotection solution was reacted twice with Leu-Ala-Arg (Pmc) -Trp-resin resin for 5 minutes and 3 minutes to obtain N-terminal Fmoc
The group was deprotected.

Next, after washing five times with DMF, washing twice with methanol and once with t-butyl methyl ether,
Further, nitrogen gas was blown to remove the remaining solvent for 10 minutes, followed by drying.

After performing the above treatment, the obtained protected peptide resin was taken out of the reaction chamber, and the livage solution (94
Mix 00 ml of trifluoroacetic acid, 300 ml of thioanisole and 300 ml of ethanedithiol,
g of 2-methylindole)
By adding l and reacting at room temperature for 2 hours, cleavage (separation) of the peptide chain from the resin resin and removal of the amino acid side chain protecting group were performed to obtain a peptide solution. To this peptide solution, 10 ml of cold ether was added to precipitate the contained peptide. Centrifuge this (2000x
g, 10 minutes) The operation of collecting the precipitate, dispersing by adding cold ether again, and centrifuging to collect was repeated four times.
The peptide was washed. The obtained peptide was lyophilized to obtain the desired peptide (I).

Similarly, peptides (II) to (VIII) having the following seven amino sequences were synthesized. Trp-His-Asn-Trp-Thr-Trp-Thr-Gly-Leu-Pro-Gly-Gln (II) Gly-His-Trp-Thr-Ala-Ser-Thr-Asn-Tyr-Val-Arg-Pro ( III) Gln-Thr-Thr-His-Trp-Asp-Ser-Ala-Lys-Tyr-Pro-Ala (IV) Phe-His-Glu-Asn-Trp-Pro-Ser (V) Phe-His-Trp- Trp-Tyr-Thr-Ile (VI) Phe-Pro-Ala-His-Ala-Ser-Arg (VII) Tyr-Ser-Phe-Glu-Arg-Ser-Lys (VIII)

Confirmation of binding ability 1 mg of the synthesized peptide (I) was added to a phosphate buffer (pH 8.0).
Dissolved to a concentration of / ml. Fluorescein isothiocyanate (FITC) was added at a molar ratio of 8 times that of peptide (I), and reacted at room temperature for 40 minutes.
For the labeling reaction, use Sephadex G-25 (50 mM
The reaction was terminated by quick loading on a column (equilibrated with a phosphate buffer (pH 7.5)). The eluted fraction labeled with FITC was collected, applied to a CM-52 column (preliminarily equilibrated with 10 mM phosphate buffer (pH 7.5)), and
Elution was performed with a linear gradient of 50 mM sodium phosphate buffer (pH 7.5), and various FITC-labeled peptides were collected. For each fraction collected, the amount of peptide was quantified from the absorbance at 280 nm, and the amount of the introduced FITC was quantified from the absorbance at 494 nm. F at a 1: 1 molar ratio to peptide
An ITC-labeled peptide fraction was prepared.

Firefly-derived luciferase (manufactured by Sigma) is dissolved in 100 mM sodium carbonate (pH 8.6) to a concentration of 0.1 mg / ml, added to a micro test well, and allowed to stand at 4 ° C. for 12 hours. Luciferase was immobilized on the wells. Discard the luciferase solution and add 5 mg / ml bovine serum albumin (Sigma) and 100 mM sodium carbonate (pH 8.6) containing 0.02% sodium azide (blocking buffer) instead.
Blocked for 1 hour at room temperature. The blocking buffer was discarded, and instead, a Tris-HCl buffer (pH 7.5) (TBST buffer) containing 0.5% Tween-20 and 150 mM sodium chloride was added, and the wells were washed well.

The FITC-labeled peptide was added to the luciferase-immobilized micro test well at various concentrations. After standing at room temperature for 1 hour, the amount of labeled peptide adsorbed on the wells by binding to luciferase was quantified from the fluorescence spectrum of FITC.

The ratio of the amount of adsorbed / unadsorbed labeled peptide to the amount of adsorbed labeled peptide is plotted (Scatchart).
d plot), a linear relationship was obtained, and a binding constant of 10 ¹⁰ [l / mol] could be obtained from the slope.

Similarly, the remaining seven peptides (II) to (V)
Regarding III), the binding constant was measured using a micro test well in which FITC was labeled and luciferase was immobilized, and a binding constant similar to that of peptide (I) was obtained. The binding constants are shown below.

[0066]

[Table 1]

(Example 2) Synthesized amino acid sequence (I)
Using eight kinds of peptides each having (VIII),
It was verified that luciferase can be detected with high quantitativeness.

Preparation of Fluorescent Labeled Luciferase Firefly-derived luciferase (manufactured by Sigma) was dissolved in phosphate buffer (pH 8.0) to a concentration of 1 mg / ml. Fluorescein isothiocyanate (FITC) was added at a molar ratio of 8 to luciferase, and reacted at room temperature for 40 minutes. For the labeling reaction, use Sephadex G-25 (50 mM phosphate buffer (pH
Termination was performed by quickly loading the column (equilibrated in 7.5). The eluted fraction labeled with FITC is collected and CM
-52 column (previously equilibrated with 10 mM phosphate buffer (pH 7.5)), eluted with a linear gradient of 10-150 mM sodium phosphate buffer (pH 7.5), and various FITC-labeled The luciferase fraction was collected. For each fraction, the amount of luciferase was quantified from the absorbance at 280 nm, and the amount of the introduced FITC was quantified from the absorbance at 494 nm. A FITC-labeled luciferase fraction was prepared at a molar ratio of 1: 1 with respect to luciferase.

Measurement of Luciferase by Competition Method Synthetic peptide (I) was added to 100 mM sodium carbonate (pH 8.6)
pmol / ml and dissolve in micro test wells
By adding 0.1 ml and leaving at 4 ° C for 12 hours,
Peptide (I) was immobilized on the wells. The peptide (I) solution was discarded, and instead, 5 mg / ml bovine serum albumin (manufactured by Sigma) and 100 mM sodium carbonate (pH 8.6) containing 0.02% sodium azide (blocking buffer) were added, and the mixture was incubated at room temperature for 1 hour. Blocked. Discard the blocking buffer and replace with 0.5% Tween-20, Tris-HCl buffer (pH 7.5) containing 150 mM sodium chloride (TBST).
Buffer) and the wells were washed well.

A fixed amount of 1 pmol of the above-mentioned FITC-labeled luciferase was added to the micro test well on which the peptide (I) was immobilized.
And 0.1 to 10 pmol of unlabeled luciferase were added. After allowing to stand at room temperature for 1 hour, the luciferase solution was taken out, the well was washed well with TBST buffer, and the amount of labeled luciferase adsorbed on the well by binding to the peptide (I) was quantified from the fluorescence amount of FITC.

A calibration curve was prepared by plotting the amount of labeled luciferase adsorbed against the amount of unlabeled luciferase charged. FIG. 1 shows an example of the created calibration curve. The fluorescence intensity on the vertical axis in the plot of FIG. 1 is 10 pmol of peptide (I), and only unlabeled luciferase was added without adding unlabeled luciferase.
The fluorescence intensity observed when adding pmol and binding
This is a relative value when 1 is set. By using such a calibration curve, 0.1 to 10 pmol of unlabeled luciferase contained in the sample could be quantified.

Similarly, the remaining seven peptides (II) to (V)
Regarding (III), each peptide was immobilized on a micro test well, and a quantitative test was carried out using FITC-labeled luciferase by a competition method for unlabeled luciferase.As for any of peptides (II) to (VIII), As a result, 0.1 to 10 pmol of luciferase contained in the sample could be quantified.

[0073]

The luciferase-binding peptide of the present invention is a peptide containing an amino acid sequence having an amino acid length of 12 or less and showing specific binding to luciferase. Such a peptide is obtained by adding a labeling substance to the peptide chain. Thus, it can be easily formed as a labeled probe, and can be easily supported and fixed on an appropriate support. By utilizing these characteristics, for example, if the sample is brought into contact with a sample containing luciferase, luciferase in the sample can be captured in the vicinity of a predetermined support via the bond with the peptide. Therefore, it is possible to detect and further quantify luciferase in a biological sample in vitro by utilizing a competitive reaction with a known concentration of luciferase provided with a labeling substance.

[0074]

[Sequence List] SEQUENCE LISTING <110> CANON INC. <120> Peptide Having Binding Affinity to Luciferase <130> 4278196 <160> 8 <170> Microsoft Word <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Selected peptide fragment based on affinity of binding to Lucifera se <400> 1 Lys His Val Asn Glu Leu Arg Glu Leu Ala Arg Trp 1 5 10 <210> 2 <211> 12 <212> PRT < 213> Artificial Sequence <220> <223> Selected peptide fragment based on affinity of binding to Luciferase <400> 2 Trp His Asn Trp Thr Trp Thr Gly Leu Pro Gly Gln 1 5 10 <210> 3 <211> 12 <212 > PRT <213> Artificial Sequence <220> <223> Selected peptide fragment based on affinity of binding to Lucifera se <400> 3 Gly His Trp Thr Ala Ser Thr Asn Tyr Val Arg Pro 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Selected peptide fragment based on affinity of binding to Lucifera se <400> 4 Gln Thr Thr His Trp Asp Ser Ala Lys Tyr Pro Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Selected peptide fragment based on affinity of binding to Lucifera se <400> 5 Phe His Glu Asn Trp Pro Ser 1 5 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Selected peptide fragment based on affinity of binding to Lucifera se <400> 6 Phe His Trp Trp Tyr Thr Ile 1 5 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Selected peptide fragment based on affinity of binding to Lucifera se <400> 7 Phe Pro Ala His Ala Ser Arg 1 5 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Selected peptide fragment based on affinity of binding to Lucifera se <400> 8 Tyr Ser Phe Glu Arg Ser Lys 1 5

[Brief description of the drawings]

FIG. 1 is an example of a quantification curve used for quantification of luciferase by a competition method using binding and immobilization with a luciferase-binding peptide of the present invention.

Claims (11)

[Claims] 1. The following eight amino acid sequences (I) to (VII)
A peptide having a luciferase-binding property, comprising a peptide chain comprising all or at least one amino acid sequence selected from the group consisting of I) or a partial sequence having an amino acid length of 5 or more. Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Leu-Ala-Arg-Trp (I) Trp-His-Asn-Trp-Thr-Trp-Thr-Gly-Leu-Pro-Gly-Gln ( II) Gly-His-Trp-Thr-Ala-Ser-Thr-Asn-Tyr-Val-Arg-Pro (III) Gln-Thr-Thr-His-Trp-Asp-Ser-Ala-Lys-Tyr-Pro- Ala (IV) Phe-His-Glu-Asn-Trp-Pro-Ser (V) Phe-His-Trp-Trp-Tyr-Thr-Ile (VI) Phe-Pro-Ala-His-Ala-Ser-Arg ( VII) Tyr-Ser-Phe-Glu-Arg-Ser-Lys (VIII) 2. A peptide having a luciferase-binding property, comprising a peptide chain comprising the entire amino acid sequence (I) or a partial sequence having an amino acid length of 5 or more. Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Leu-Ala-Arg-Trp (I) 3. A peptide having a luciferase-binding property, comprising a peptide chain comprising the entire amino acid sequence (II) or a partial sequence having an amino acid length of 5 or more. Trp-His-Asn-Trp-Thr-Trp-Thr-Gly-Leu-Pro-Gly-Gln (II) 4. A peptide having a binding property to luciferase, comprising a peptide chain comprising the whole of the following amino acid sequence (III) or a partial sequence having an amino acid length of 5 or more. Gly-His-Trp-Thr-Ala-Ser-Thr-Asn-Tyr-Val-Arg-Pro (III) 5. A peptide having a luciferase-binding property, comprising a peptide chain comprising the entire amino acid sequence (IV) or a partial sequence having an amino acid length of 5 or more. Gln-Thr-Thr-His-Trp-Asp-Ser-Ala-Lys-Tyr-Pro-Ala (IV) 6. A peptide having a binding property to luciferase, comprising a peptide chain comprising the whole of the following amino acid sequence (V) or a partial sequence having an amino acid length of 5 or more. Phe-His-Glu-Asn-Trp-Pro-Ser (V) 7. A peptide having a luciferase-binding property, comprising a peptide chain comprising the entire amino acid sequence (VI) or a partial sequence having an amino acid length of 5 or more. Phe-His-Trp-Trp-Tyr-Thr-Ile (VI) 8. A peptide having a luciferase-binding property, comprising a peptide chain comprising the entire amino acid sequence (VII) below or a partial sequence having an amino acid length of 5 or more. Phe-Pro-Ala-His-Ala-Ser-Arg (VII) 9. A peptide having a luciferase-binding property, comprising a peptide chain comprising the entire amino acid sequence (VIII) or a partial sequence having an amino acid length of 5 or more. Tyr-Ser-Phe-Glu-Arg-Ser-Lys (VIII) 10. As a means for detecting luciferase, all of at least one kind of amino acid sequence selected from the group consisting of the following eight kinds of amino acid sequences (I) to (VIII), or a portion having an amino acid length of 5 or more: A luciferase detection kit comprising a peptide having a binding property to luciferase, comprising a peptide chain comprising a sequence. Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Leu-Ala-Arg-Trp (I) Trp-His-Asn-Trp-Thr-Trp-Thr-Gly-Leu-Pro-Gly-Gln ( II) Gly-His-Trp-Thr-Ala-Ser-Thr-Asn-Tyr-Val-Arg-Pro (III) Gln-Thr-Thr-His-Trp-Asp-Ser-Ala-Lys-Tyr-Pro- Ala (IV) Phe-His-Glu-Asn-Trp-Pro-Ser (V) Phe-His-Trp-Trp-Tyr-Thr-Ile (VI) Phe-Pro-Ala-His-Ala-Ser-Arg ( VII) Tyr-Ser-Phe-Glu-Arg-Ser-Lys (VIII) 11. The following eight amino acid sequences (I) to (VI)
II) binding to luciferase, which comprises at least one kind of amino acid sequence selected from the group consisting of, or a partial sequence having an amino acid length of 5 or more thereof and a peptide chain having a total amino acid length of 30 or less A peptide having the property. Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Leu-Ala-Arg-Trp (I) Trp-His-Asn-Trp-Thr-Trp-Thr-Gly-Leu-Pro-Gly-Gln ( II) Gly-His-Trp-Thr-Ala-Ser-Thr-Asn-Tyr-Val-Arg-Pro (III) Gln-Thr-Thr-His-Trp-Asp-Ser-Ala-Lys-Tyr-Pro- Ala (IV) Phe-His-Glu-Asn-Trp-Pro-Ser (V) Phe-His-Trp-Trp-Tyr-Thr-Ile (VI) Phe-Pro-Ala-His-Ala-Ser-Arg ( VII) Tyr-Ser-Phe-Glu-Arg-Ser-Lys (VIII)