JP4618751B2 - Peptide having binding property to luciferase - Google Patents

Description

【００６７】
（実施例２）
合成したアミノ酸配列(I)〜(VIII)をそれぞれ有する８種類のペプチドを利用して、ルシフェラーゼを高い定量性で検出できることを検証した。
【００６８】
蛍光標識ルシフェラーゼの調製
ホタル由来のルシフェラーゼ（シグマ社製）をリン酸バッファー(pH8.0)に1 mg/mlの濃度に成るように溶解した。フルオレセイン イソチオシアネート(FITC)を、ルシフェラーゼに対してモル比で8倍になるように添加し、室温で40分間反応させた。標識反応は、反応溶液をセファデックスG-25（予め50mMのリン酸バッファー(pH7.5)で平衡化した）カラムに素早くロードすることによって終結させた。FITC標識された溶出画分を分取し、CM-52カラム（予め10mMのリン酸バッファー(pH7.5)で平衡化した）にアプライし、10〜150mMのリン酸ナトリウムバッファー(pH7.5)の直線グラジエントで溶出し、様々なFITC標識されたルシフェラーゼ画分を分取した。各画分について、280nmにおける吸光度からルシフェラーゼ量を定量し、494nm吸光度から導入されたFITC量を定量した。ルシフェラーゼに対して、1:1のモル比でFITC標識されたルシフェラーゼ画分を調製した。
【００６９】
競合法によるルシフェラーゼの測定
合成ペプチド(I)を100mM 炭酸ナトリウム(pH8.6)に100 pmol/mlになるように溶解させ、マイクロ試験ウエルに0.1 ml添加し、４℃で１２時間放置することによって、ペプチド(I)をウエル上に固定化した。ペプチド(I)溶液を棄て、代わりに5 mg/ml ウシ血清アルブミン（シグマ社製）、0.02％アジ化ナトリウムを含む100mM 炭酸ナトリウム(pH8.6)（ブロッキング・バッファー）を加え、室温で１時間ブロッキングした。ブロッキング・バッファーを棄て、代わりに0.5％Tween-20、150mM 塩化ナトリウムを含むトリス−塩酸バッファー(pH7.5)(TBSTバッファー)を加え、ウエルを良く洗浄した。
【００７０】
ペプチド(I)を固定化したマイクロ試験ウエルに、上記FITC標識ルシフェラーゼの一定量1 pmolと、標識していないルシフェラーゼの0.1 〜 10 pmolとを添加した。室温で１時間放置後、ルシフェラーゼ溶液を取り出し、ウエルをTBSTバッファーで良く洗浄し、ペプチド(I)との結合により、ウエル上に吸着された標識ルシフェラーゼをFITCの蛍光量から定量した。
【００７１】
投入した標識していないルシフェラーゼ量に対して、吸着された標識ルシフェラーゼの量をプロットすることによって、検量線を作成した。図1に、作成された検量線の一例を示す。図1のプロットにおける縦軸の蛍光強度は、10 pmolのペプチド(I)に対して、非標識ルシフェラーゼを加えずに標識ルシフェラーゼのみ 1 pmolを添加し、結合させた際に、観測される蛍光強度を1としたときの相対値である。このような検量線を利用することによって、試料中に含まれる0.1 〜 10 pmolの非標識ルシフェラーゼを定量することができた。
【００７２】
同様にして、残る７種のペプチド(II)〜(VIII)に関しても、各ペプチドをマイクロ試験ウエルに固定化し、FITC標識したルシフェラーゼを用いて、標識されていないルシフェラーゼの競合法による定量試験を行ったところ、ペプチド(II)〜(VIII)のいずれについても、試料中に含まれる0.1 〜 10 pmolのルシフェラーゼを定量することができた。
【００７３】
【発明の効果】
本発明のルシフェラーゼ結合性ペプチドは、ルシフェラーゼに対する特異的な結合性を示すアミノ酸長が１２以下のアミノ酸配列を含むペプチドであり、かかるペプチドは、そのペプチド鎖に標識物質を付加して、標識プローブとすること、また、然るべき支持体上に担持・固定することが容易にできる。これらの特徴を利用して、例えば、ルシフェラーゼを含む試料と接触させれば、試料中のルシフェラーゼをかかるペプチドとの結合を介して、所定の支持体近傍に捕捉することができる。従って、標識物質を備えた既知濃度のルシフェラーゼとの競合反応などの利用により、生物学的試料におけるインビトロでのルシフェラーゼを検出、さらには、定量をすることが可能となる。
【００７４】
【配列表】

【図面の簡単な説明】
【図１】本発明のルシフェラーゼ結合性ペプチドによる結合・固定を利用した競合法によるルシフェラーゼの定量に用いる定量曲線の一例である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a peptide containing an amino sequence having binding property to luciferase, and a luciferase detection kit using the peptide having binding property to luciferase as a detection means.
[0002]
[Prior art]
Luciferase is a general term for oxygenases that catalyze bioluminescence, and is also called a luminescent enzyme. This enzyme catalyzes the oxidation of luciferin by oxygen molecules, and chemiluminescence derived from the substrate luciferin is obtained with the chemical change at that time. The reaction mechanism is known to vary depending on the species originating from luciferase. Of these, the bioluminescence reaction by luciferase isolated from fireflies is the most studied. Firefly luciferase (EC.1.13.12.7) acts on adenosine triphosphate (ATP) and a luciferase substrate typified by firefly luciferin to produce adenosine monophosphate (AMP) and oxyluciferin and produce luminescence at the same time.
[0003]
The bioluminescence reaction by luciferase is very useful as an analytical method because it is advantageous in terms of sensitivity compared to chemiluminescence using peroxidase. The most typical application field of luciferase is as an analytical reagent. Specific examples of the analysis reagent include firstly various binding analysis systems using luciferase as a labeling substance. The binding analysis system includes an immunoassay utilizing an antigen-antibody reaction, a nucleic acid hybridization assay utilizing the affinity of complementary nucleic acids, and avidin-biotin, hormone-hormone receptor, and sugar-lectin. Analytical systems using the binding between substances having such specific affinity are known. In these binding analysis systems, if luciferase is used as a labeling substance (labeling enzyme), the amount of the substance finally bound (or not bound) in the binding analysis system by measuring the enzyme activity. 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 for the enzyme reaction is also conceivable. For example, when screening a foreign gene using a luciferase gene as an index or analyzing a gene mutation, it is necessary to track the activity of the luciferase produced.
[0005]
The ATP detection method using firefly luciferase is used for the measurement of the number of cells, the tracking of enzyme reaction using ATP as an enzyme reaction product as an index, and the binding analysis system using ATP itself as a label. ing. 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, a drug It has been reported that it can be applied to measure the degree of influence of stress such as.
[0006]
On the other hand, the luciferase itself is generally quantified based on its function and enzyme activity. In JP 07-069899 A, the common logarithm of the luciferase concentration and the common logarithm of the enzyme activity (integrated value of fluorescence) are placed on a straight line as a linear function. It is disclosed that it is possible. For example, for luciferase expressed in infected cells, the activity value (integrated value of fluorescence) is measured, and the protein amount is determined based on the calibration curve.
[0007]
In addition, in Japanese Patent Application Laid-Open No. 08-116991, the amount of luciferase contained in the body of a transgenic nematode is determined using the fact that the amount of luminescence depends on the amount of luciferase added under certain conditions. Measured as an increase in the amount of luminescence.
[0008]
[Problems to be solved by the invention]
In the method for quantifying luciferase based on its enzyme activity (amount of luminescence), it is assumed that the conditions for preparing a calibration curve are the same as the conditions for sample measurement. However, it is often difficult to guarantee this precondition when trying to quantify the amount of luciferase in vivo, for example in recombinants. In addition, luciferase extracted from biological materials such as fireflies has a problem in its stability and its enzymatic activity decreases over time, so when using a functional quantification method based on the amount of luminescence, the measurement data There is a limit to its reliability.
[0009]
Therefore, in place of the method of quantifying based on the enzyme activity (the amount of luminescence), a method of quantifying luciferase, more specifically, a method of quantifying luciferase using a peptide having binding property to luciferase as a detection means Suggestion is desired.
[0010]
The present invention solves the above-mentioned problems, and an object of the present invention is to provide a peptide having a binding property to luciferase, which can be used for direct quantification of luciferase, and a peptide having a binding property to such luciferase. Is to provide a detection method for luciferase, and a detection kit exclusively used for it. In addition, the peptide which has the binding property with respect to luciferase which is the object of the present invention is not only a direct quantification of luciferase in a sample but also a useful means for separating and purifying luciferase.
[0011]
[Means for Solving the Problems]
In order to solve the above problems, the present inventor has screened peptides having a specific binding property to luciferase from a random peptide library. As a result, a total of 8 types of peptides have sufficiently high binding. It was found to have the ability. For these 8 peptides, the amino acid sequences were analyzed, and peptide chains thought to be involved in binding to luciferase were identified. Specifically, it was found that the following peptide chains (I) to (VIII) have high binding properties to luciferase.
Peptide (I): Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Leu-Ala-Arg-Trp
Peptide (II): Trp-His-Asn-Trp-Thr-Trp-Thr-Gly-Leu-Pro-Gly-Gln
Peptide (III): Gly-His-Trp-Thr-Ala-Ser-Thr-Asn-Tyr-Val-Arg-Pro
Peptide (IV): Gln-Thr-Thr-His-Trp-Asp-Ser-Ala-Lys-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 were compared with each other, there was no significant homology among any of the peptides (I) to (VIII), and the specific binding properties to luciferase were as follows. It was judged to be achieved by the partial sequence contained in the amino acid sequence. The present inventor has completed the present invention based on such knowledge. Each has a different amino acid sequence.
[0012]
That is, the peptide having binding property to the luciferase of the present invention is:
For luciferase comprising a peptide chain comprising the whole of at least one amino acid sequence selected from the group consisting of the following 8 amino acid sequences (I) to (VIII) or a partial sequence having an amino acid length of 5 or more: A peptide having binding properties.
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)
[0013]
For example, a peptide having a binding property to the luciferase of the present invention is:
Respectively,
The following amino acid sequence (I):
Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Leu-Ala-Arg-Trp (I)
A peptide comprising a peptide chain comprising a partial sequence of 5 or more of its amino acid length,
The following amino acid sequence (II):
Trp-His-Asn-Trp-Thr-Trp-Thr-Gly-Leu-Pro-Gly-Gln (II)
A peptide comprising a peptide chain comprising a partial sequence of 5 or more of its amino acid length,
The following amino acid sequence (III):
Gly-His-Trp-Thr-Ala-Ser-Thr-Asn-Tyr-Val-Arg-Pro (III)
A peptide comprising a peptide chain comprising a partial sequence of 5 or more of its amino acid length,
The following amino acid sequence (IV):
Gln-Thr-Thr-His-Trp-Asp-Ser-Ala-Lys-Tyr-Pro-Ala (IV)
A peptide comprising a peptide chain comprising a partial sequence of 5 or more of its amino acid length,
The following amino acid sequence (V):
Phe-His-Glu-Asn-Trp-Pro-Ser (V)
A peptide comprising a peptide chain comprising a partial sequence of 5 or more of its amino acid length,
The following amino acid sequence (VI):
Phe-His-Trp-Trp-Tyr-Thr-Ile (VI)
A peptide comprising a peptide chain comprising a partial sequence of 5 or more of its amino acid length,
The following amino acid sequence (VII):
Phe-Pro-Ala-His-Ala-Ser-Arg (VII)
A peptide comprising a peptide chain comprising a partial sequence of 5 or more of its amino acid length,
Or
The following amino acid sequence (VIII):
Tyr-Ser-Phe-Glu-Arg-Ser-Lys (VIII)
Or a peptide consisting of a peptide chain comprising a partial sequence of 5 or more amino acids in length.
[0014]
In addition, the present invention also provides a method of using the above-mentioned peptide having binding property to luciferase for detection of luciferase, and also provides a kit used for this detection method. That is, the luciferase detection kit of the present invention comprises
As a means for detecting luciferase,
For luciferase comprising a peptide chain comprising the whole of at least one amino acid sequence selected from the group consisting of the following 8 amino acid sequences (I) to (VIII) or a partial sequence having an amino acid length of 5 or more: A luciferase detection kit comprising a binding peptide.
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 participating in the composition of the peptide according to the present invention, NUCLEIC ACIDS RESERCH 13, 3021-3030 (1985) and THE Biological Journal (THE BIOLOGICAL JOURNAL) The IUPAC-IUB rules (Rules) shown in 219, No. 2, 345-371 (1984) are used.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the peptide having the binding property to the luciferase of the present invention, the preparation method thereof, and the method of using the luciferase-binding peptide in the luciferase detection kit of the present invention will be described in more detail.
[0017]
The peptide having binding ability to the luciferase of the present invention is composed of at least one of the above-mentioned eight kinds of amino acid sequences (I) to (VIII) or a peptide chain containing a partial sequence having an amino acid length of 5 or more. ing. That is, the amino acid sequence exhibiting the binding property to the luciferase may be present as a peptide containing at least a part of the peptide chain constituting the amino acid sequence. When such a peptide chain containing the amino acid sequence exhibiting binding property to luciferase as a partial sequence thereof is used, any remaining amino acid sequence may be adopted as long as the binding property to luciferase is not inhibited. Also good.
[0018]
The luciferase-binding peptide according to the present invention can be prepared according to its entire amino acid sequence by any known organic chemical peptide synthesis method or using recombinant DNA technology.
[0019]
The organic chemical peptide synthesis method includes binding necessary amino acids together in a homogeneous phase or by a condensation reaction using a so-called solid phase.
[0020]
The condensation reaction used for peptide chain elongation is:
a) Compounds having a free carboxyl group and other reactive groups are protected (amino acids, peptides), compounds having a free amino group and other reactive groups are protected (amino acids, peptides) And condensing in the presence of a condensing agent, or
b) Activated carboxyl group, other reactive groups are free or protected compounds (amino acids, peptides), have free amino groups, other reactive groups are free Or condensing with protected compounds (amino acids, peptides),
It can be carried out by using the method a) or b) described above.
[0021]
In the above-mentioned method b), the activation of the carboxyl group is carried out in particular by converting the carboxyl group into an acid halide, azide, acid anhydride, imidazolide, or N-hydroxy-succinimide, N-hydroxy-benzotriazole or p-nitrophenyl. By converting to an activated ester such as an ester.
[0022]
Among these, the most common methods for causing the condensation reaction are the carbodiimide method, the azide method, the mixed acid anhydride method, and E.I. Gross and J.M. Edited by Meienhofer, The Peptides, Analysis, Synthesis, Biology, vols. 1-3, Academic Press Inc. , 1979, 1980, 1981, using activated esters.
[0023]
Peptide synthesis methods that can be used when preparing appropriate fragments of the above-described peptides according to the present invention using the “solid phase method” include, for example, J. Amer. Chem. Soc. 85, p. 2149, 1963 and Int. J. et al. Peptide Protein Res. 35, pp. 161-214, 1990. In this “solid phase method”, the amino acid bond (elongation of the peptide chain) of the peptide to be prepared usually starts 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. As the solid phase, for example, a copolymer of benzene and divinylbenzene and a reactive chloromethyl group, or a polymer solid phase imparted with a property of reacting with a hydroxymethyl or amine functional group can be used. Particularly suitable solid phases are described, for example, in J. Org. Am. Chem. Soc. 95, p. 1328, 1974, p-alkoxybenzyl alcohol resin (4-hydroxy-methyl-phenoxy-methyl-copolystyrene-1% divinylbenzene resin) described by Wang.
[0024]
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 also be separated from the support by transesterification using a lower alcohol, preferably methanol or ethanol, in which case the separated peptide is in the form of a lower alkyl ester. Similarly, the peptide can be separated using ammonia, and in this case, the separated peptide is in the form of an amide at its C-terminus.
[0025]
As described above, the reactive group that does not participate in the above condensation reaction (elongation of peptide chain), specifically, the reactive group on the amino acid residue and the amino group at the N-terminal of the peptide chain, It is effectively protected by a group which can be removed again very easily by hydrolysis or reduction with. For example, a carboxyl group can be effectively protected by, for example, esterification with methanol, ethanol, t-butanol, benzyl alcohol or p-nitrobenzyl alcohol, and an amine bound to a solid support. On the other hand, the group capable of effectively protecting the amino group is ethoxycarbonyl, benzyloxycarbonyl, t-butoxy-carbonyl (t-boc) or p-methoxy-benzyloxycarbonyl group, or benzene-sulfonyl or p- Acid groups derived from sulfonic acids, such as toluene-sulfonyl groups, but substituted or unsubstituted aryl or aralkyl groups such as benzyl and triphenylmethyl, ortho-nitrophenyl-sulfenyl and 2-benzoyl-1-methyl It is also possible to use other groups such as groups such as vinyl. In particular, suitable α-amino protecting groups are, for example, the base-sensitive 9-fluorenyl-methoxycarbonyl (Fmoc) group (Carpino and Han, J. Amer. Chem. Soc. 92, p. 5748, 1970).
[0026]
A more extensive description of protecting groups that can be used in peptide synthesis can be found in Gross, Udenfriend and Meienhofer, The Peptides, Analysis, Synthesis, Biology, vols. 1-9, Academic Press Inc. , 1979-1987.
[0027]
For example, in addition to the α-amino group, it is necessary to protect the ε-amino group of lysine, and it is appropriate to protect the guanidine group of arginine. The protecting group usually used for such protection is Boc group in the case of lysine, and Pmc group, Pms group, Mbs group or Mtr group in the case of arginine.
[0028]
These protecting groups depend on the nature of the group in various conventional ways, for example with trifluoroacetic acid or by mild reduction with hydrogen and a catalyst such as palladium, or with HBr in glacial acetic acid. And can be removed.
[0029]
The luciferase-binding peptide according to the present invention may be a single molecule that is a series of amino acid sequences by combining a plurality of amino acid sequences having binding properties with luciferase therein. A hybrid peptide or a complex peptide corresponding to the two or more partial peptide chains linked by a covalent bond can be obtained, for example, by using the above-described method according to a target series of amino acid sequences. This is possible by solid phase peptide synthesis. At that time, the amino acid sequences corresponding to the individual partial peptide chains are aligned with each other. A linker sequence can also be inserted between amino acid sequences corresponding to individual partial peptide chains. Such linker sequences can also utilize, for example, a 2-5 residue stretch of glycine.
[0030]
Furthermore, hybrid or complex peptides can be prepared by solid phase synthesis using fragment condensation techniques. In advance, a plurality of fragments each having a predetermined amino acid sequence, including fragments (partial peptide chains) whose amino acid sequences include any of the amino acid sequences (I) to (VIII) above, are 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 approach is preferred when synthesizing relatively long hybrid or complex peptide sequences. Methods for preparing relatively long peptides are known to those skilled in the art and are described, for example, in The Peptides, Analysis, Synthesis, Biology, vols. 1-9 (see above).
[0031]
Alternatively, in addition to the above-described series of long peptide chains, the luciferase-binding peptide of the present invention can also be prepared as a hybrid or complex peptide by binding appropriately modified peptide chains of the present invention.
[0032]
For example, when the peptide chain to be bound itself does not contain a cysteine amino acid in its amino acid sequence, one preferred method for joining these two different peptide chains is to link each peptide chain to its carboxyl terminus. Alternatively, it is a method of converting to a derivative having an additional cysteine residue at the amino terminus and forming a disulfide bond between the additional cysteine residues. In this method of forming a disulfide bond, after conversion to a derivative having a cysteine residue at the terminal, the thiol functional group of the introduced single cysteine is converted to 2,2′-dithio for one peptide derivative. Activate with dipyridine. The obtained pyridyl-dithio-peptide derivative is reacted with a second peptide derivative having a cysteine thiol group to obtain a hybrid peptide in which individual peptide chains are linked by a disulfide bond.
[0033]
Various other mixed peptide preparation methods are also available. For example, chemical methods developed in the field of protein-protein binding can also be used. Refer to Means and Feeney (Bioconj. Chem. 1, pp. 2-12, 1990) for such a method of linking between peptide chains. For example, when a well-known homo- or heterobifunctional cross-linking agent is used, individual peptide chains can be linked by a disulfide bond, a thioether bond, an amide bond, or the like.
[0034]
After 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 other types of chromatography to give a 95% pure peptide in a purity assay by analytical HPLC.
[0035]
As mentioned earlier, the luciferase binding peptides according to the present invention can also be prepared using recombinant DNA technology. The availability of this recombinant DNA technology can be achieved by incorporating a partial peptide chain of the desired amino acid sequence “in tandem” into a repetitive sequence, or by incorporating a partial peptide chain in a protein or polypeptide having a much larger total amino acid length. When prepared as a component or as a fusion protein with a general-purpose fusion partner such as (part of) β-galactosidase, or replaced with the amino acid sequence of the hypervariable region in another antibody This is particularly important when creating artificial antibodies. Therefore, as described above, the main portion 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, any one of the above eight amino acid sequences (I) to (VIII) or a partial sequence having an amino acid length of 5 or more, which characterizes the luciferase-binding peptide according to the present invention as a component of recombinant DNA A nucleic acid sequence encoding a partial peptide chain containing is used.
[0036]
In the method using the recombinant DNA technique, a desired peptide is prepared by expressing a recombinant polynucleotide containing a nucleic acid sequence encoding one or more luciferase-binding peptide chain portions to be prepared in a suitable microorganism as a host. including.
[0037]
The nucleic acid sequence encoding the luciferase-binding peptide chain portion according to the present invention is inserted as a DNA fragment having the base sequence into a commonly used plasmid or the like, and ligated with various DNA sequences that realize replication. , So-called recombinant vector molecules. This recombinant vector molecule can be used to transform a suitable host. Useful recombinant vector molecules are preferably derived from, for example, plasmids, bacteriophages, cosmids or viruses.
[0038]
Specific vectors or cloning vehicles that can be used to clone the nucleic acid sequence of interest are known to those skilled in the art, in particular plasmid vectors such as pBR322, various pUC, pGEM and Bluescript plasmids, bacteriophages such as kgt-Wes, Charon 28 and M13-derived phages, as well as viral vectors such as SV40, adenovirus or polyoma virus (edited by RL Rodriquez and DT Denhardt, Vectors: A survey of molecular cloning vectors) J. A. Lenstra et al., Arch. Virol. 110, pp. 1-, Butterworths, 1988. 4, 1990 see also). Techniques to be used for the construction of recombinant vector molecules are known to those skilled in the art, Maniatis et al. (Molecular Cloning: Laboratory Manual, second edition, Cold Spring Harbor Laboratory, 1989).
[0039]
For example, inserting a nucleic acid sequence (DNA fragment) encoding a peptide into the luciferase-binding peptide chain portion according to the present invention into a cloning vector means that one or more ends of the inserted gene (DNA) may be inserted as necessary. Cleavage using a restriction enzyme, and the cloning site (site) of the desired cloning vehicle is also digested with the same restriction enzyme to form complementary DNA ends at the ends to be joined to each other. Thus, insertion and connection can be easily performed.
[0040]
It should be noted that the recombinant vector molecule is one or more kinds that can be used for selection of a desired transformant such as pBR322 ampicillin and tetracycline resistance, and pUC8 ampicillin resistance and β-galactosidase α-peptide coding region activity. May additionally have the marker activity (gene).
[0041]
A luciferase-binding peptide according to the present invention is modified in vivo or in vitro on its peptide chain, for example by glycosylation, amidation, carboxylation or phosphorylation, as long as the amino acid sequence conferring luciferase binding is maintained. It is also possible to do. Accordingly, examples of the luciferase-binding peptide according to the present invention include functional modifications such as acid addition salts, amides, esters, particularly C-terminal esters, and N-acyl derivatives, as part of the present invention. .
[0042]
Also in the luciferase-binding peptide according to the present invention, for example, when the partial peptide chain having the target amino acid sequence is included as a component of a natural protein or polypeptide as described above, It is understood that there may be natural modifications. Such modifications can be manifested by one or more amino acid differences (substitutions) found in the entire amino acid sequence, or by deletion or insertion of one or more amino acids. In the meantime, various types of amino acid substitutions that are considered not to substantially change the biological activity and immune activity in natural proteins and polypeptides have been disclosed. More specifically, representative amino acid substitutions between related amino acids, or substitutions frequently occurring in the course of evolution are, for example, Ser / Ala, Ser / Gly, Asp / Gly, Asp / Asn, Ile / Val (see M. D. Dayhof, “Atlas of protein sequence and structure,” Nat. Biomed. Res. Found., Vol. 5, suppl. 3, Washington D. C. 78). For example, based on this information, Lipman and Pearson have developed a rapid and sensitive protein comparison method (Science 227, pp. 1434-1441, 1985) and measured the functional similarity between homologous proteins. In the present invention, as long as the luciferase binding property is not impaired, mutations frequently found in the natural world can also be included.
[0043]
The luciferase-binding peptide of the present invention has a specific binding property to luciferase in spite of a relatively short peptide chain. Therefore, the luciferase-binding peptide can be easily prepared and immobilized on a substrate, for example, on a substrate. Has the advantage of being easy. If this 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 through binding to the luciferase-binding peptide. it can. Thus, although the upper limit of the amino acid length of the luciferase-binding peptide of the present invention is not particularly limited, when used as an immobilized probe, the amino acid length is 50 or less, more preferably 30 or less. It is desirable to do. That is, if a peptide having a shorter chain length that 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 in the vicinity of the support. Can do. Therefore, if a competitive reaction with a known concentration of luciferase with a labeling substance, or a sandwich or agglutination reaction with a free peptide (labeled probe) with a labeling substance is used, in vitro in a biological sample. It can be used for highly sensitive detection and quantification of luciferase.
[0044]
Supports that can be used in carrying the luciferase binding peptides of the present invention include, for example, micro test wells or inner walls of cuvettes, tubes or tubules, membranes, filters, test strips, and particles such as latex particles, Aldehyde particles; specifically, surfaces such as ceramic magnetizable particles having active aldehyde surface groups, dye sols, metal sols or metal compounds as sol particles, carrier proteins such as bovine serum albumin, and the like. On the other hand, labeling substances that can be used in the above detection / quantification methods are, among others, fluorescent compounds, radioisotopes, labeling enzymes, dye sols, metal sols, or metal compounds as sol particles.
[0045]
The binding reaction between the luciferase-binding peptide of the present invention and luciferase is usually carried out at a temperature of 2 to 40 ° C., preferably 4 to 10 ° C. in a suitable buffer solution having a pH of 5 to 9, preferably about 6 to 7.5. 10 minutes to 24 hours, preferably 30 minutes to 60 minutes. The buffer solution to be used is not particularly limited. For example, a phosphate buffer solution having a concentration of about 0.01 to 1M, preferably about 0.1 to 0.5M, or a Tris-HCl buffer solution, which is widely used in the above pH range, is suitable. is there.
[0046]
After the reaction for forming the binding complex of the luciferase-binding peptide of the present invention supported on the support and the luciferase and / or after the reaction of the formed binding complex and the labeled probe, the reaction product is appropriately mixed. Unreacted substances are removed by washing with a washing solution such as distilled water, physiological saline, phosphate buffer, Triton X-100-containing buffer or Tween 20-containing buffer. The remaining bound complex is washed, and then, depending on the label, the measurement apparatus suitable for the evaluation of the enzyme activity, radioactivity, fluorescence, etc. of the labeled enzyme, for example, a spectrophotometer or well for measuring enzyme reaction products 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.
[0047]
The labeled probe used for the sandwich reaction with the free peptide (labeled probe) provided with the labeling substance described above is a peptide that selectively binds to the luciferase constituting the binding complex, that is, the present invention. The luciferase-binding peptide is used. At that time, the luciferase-binding peptide carried on the support used for the formation of the binding complex and the luciferase-binding peptide used for the labeled probe are selected so that their binding sites do not compete with each other. To do. Therefore, the two types of luciferase-binding peptides are selected so that the amino acid sequences involved in the luciferase-binding property are different from each other.
[0048]
For example, the luciferase-binding peptide supported on the support has an additional peptide part used for supporting or fixing on the support in addition to the amino acid sequence part involved in the luciferase binding property. Can be included. For example, by adding cysteine to the N-terminus or C-terminus, a monolayer of a peptide can be formed autonomously by utilizing the binding ability of the added cysteine side chain to the SH surface of the SH group. . On the other hand, the luciferase-binding peptide used for the labeling probe can also contain an additional peptide part used when attaching the labeling substance, if necessary, in addition to the amino acid sequence part involved in the luciferase binding property. .
[0049]
The luciferase detection kit of the present invention has a luciferase-binding peptide carried on a support and a standard luciferase provided with a labeling substance or a free luciferase-binding ability provided with a labeling substance, depending on the detection method exemplified above. It can be set as the kit comprised from a peptide (labeled probe).
[0050]
【Example】
Hereinafter, the present invention will be described more specifically 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.
[0051]
Example 1
Eight types of peptides each having the amino acid sequences (I) to (VIII) were chemically synthesized, and their binding properties to luciferase were verified.
[0052]
Peptide synthesis
Peptides were chemically synthesized by a method (solid phase synthesis method) in which amino acids were bonded to the amino acid derivative fixed on the resin one by one from the carboxyl terminal side. As the amino acid used in each cycle, a special α-amino acid derivative in which the reactive group of the α-amino group and the amino acid residue part (side chain) was blocked with a protecting group was used. In this example, each α-amino group was a derivative of each amino acid blocked with an Fmoc group (9-fluorenyl / methyloxycarbonyl group) (Fmoc method). In peptide synthesis (elongation of peptide chain), the Fmoc group of the α-amino group of the amino acid bonded to the resin is deprotected, and then the amino acid derivative in which the carboxyl group is activated is converted into the deprotected α-amino group. The reaction of bonding to the group was repeated sequentially. Each peptide was synthesized by the above Fmoc solid phase synthesis method using a peptide synthesizer (PSSM-8: manufactured by Shimadzu Corporation).
[0053]
That is, in the synthesis of peptide (I) having the following amino acid sequence,
Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Leu-Ala-Arg-Trp (I)
30 mg of Fmoc-Trp-resin resin (0.44 mmol / g; manufactured by Shimadzu Corporation) into which an amino acid (Trp) corresponding to the C-terminal residue of the peptide to be synthesized has been introduced is reacted with the above peptide synthesizer. It was set in a container 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 5 times with DMF. Washed. Activator solution (0.5M 1-hydroxybenzotriazole (HOBt) / DMF and 1M N-methyl) was added to 150 μmol Fmoc-Arg (Pmc) -OH / PyBOP corresponding to the second amino acid (Arg) from the C-terminal side. (Morpholine / DMF) was added and activated, and the amino acid (Trp) bound to the resin excluding the Fmoc group was reacted at room temperature for 30 minutes. Here, in order to remove the reaction solution containing unreacted Fmoc-Arg (Pmc) -OH / PyBOP and the like, the produced Fmoc-Arg (Pmc) -Trp-resin resin was washed four times with DMF. Thereafter, the Fmoc-Arg (Pmc) -Trp-resin resin was again subjected to deprotection of the N-terminal Fmoc group under the above-mentioned conditions, washed 5 times with DMF, and then activated Fmoc-Ala with an activator solution. Reacted with -OH / PyBOP solution. After completion of the reaction, the reaction solution containing unreacted Fmoc-Ala-OH / PyBOP and the like was washed four times with DMF in order to remove the reaction solution.
[0054]
Subsequent peptide chain elongation can be achieved by repeating the same procedure to obtain the desired protected peptide resin: Fmoc-Lys (Boc) -His (Trt) -Val-Asn (Trt) -Glu (OtBu) -Leu-Arg (Pmc) -Glu (OtBu) -Leu-Ala-Arg (Pmc) -Trp-resin resin was synthesized.
[0055]
In addition, the amino acid derivatives used in the peptide synthesis below this example are as follows (inside () attached to each amino acid represents a protecting group for protecting the reactive group of the amino acid residue part (side chain); (Shimadzu Corporation):
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.
[0056]
In addition, the meaning of the abbreviation used in the said description is as follows:
Trt = Trityl;
Pmc = 2,2,5,7,8-pentamethylchroman-6-sulfonyl;
Boc = t-butoxycarbonyl;
tBu = tert-butyl;
PyBOP = benzotriazol-1-yl-oxy-tris (pyrrolidino) phosphonium hexafluorophosphate;
OtBu = tert-butoxy.
[0057]
Protected peptide resin synthesized after completing a series of peptide chain elongation: Fmoc-Lys (Boc) -His (Trt) -Val-Asn (Trt) -Glu (OtBu) -Leu-Arg (Pmc) The N-terminal Fmoc group was deprotected by reacting -Glu (OtBu) -Leu-Ala-Arg- (Pmc) -Trp-resin resin with the deprotection solution twice for 5 minutes and 3 minutes.
[0058]
Next, after washing 5 times with DMF, it was washed twice with methanol and once with t-butyl methyl ether, and further nitrogen gas was blown to remove the remaining solvent and dry for 10 minutes.
[0059]
After the above treatment, the protected peptide resin obtained from the reaction chamber was taken out, and the cleaved solution (9400 ml of trifluoroacetic acid, 300 ml of thioanisole and 300 ml of ethanedithiol was mixed to dissolve 5 mg of 2-methylindole. 0.5 ml of the product) was added and reacted at room temperature for 2 hours to cleave (separate) the peptide chain from the resin resin and remove the amino acid side chain protecting group to obtain a peptide solution. To this peptide solution, 10 ml of cold ether was added to precipitate the contained peptide. The peptide was washed by centrifuging (2000 × g, 10 minutes), collecting precipitates, adding cold ether again to disperse, centrifuging and collecting the precipitates four times. The obtained peptide was lyophilized to obtain the target peptide (I).
[0060]
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)
[0061]
Check connectivity
The synthesized peptide (I) was dissolved in a phosphate buffer (pH 8.0) to a concentration of 1 mg / ml. Fluorescein isothiocyanate (FITC) was added at a molar ratio of 8 times that of peptide (I) and allowed to react at room temperature for 40 minutes. The labeling reaction was terminated by quickly loading the reaction solution onto a Sephadex G-25 (previously equilibrated with 50 mM phosphate buffer (pH 7.5)) column. The FITC-labeled elution fraction was collected, applied to a CM-52 column (previously equilibrated with 10 mM phosphate buffer (pH 7.5)), and 10 to 150 mM sodium phosphate buffer (pH 7.5). And eluted with a linear gradient of various FITC-labeled peptides. For each fraction, the amount of peptide was quantified from the absorbance at 280 nm, and the amount of FITC introduced was quantified from the absorbance at 494 nm. A peptide fraction labeled with FITC was prepared at a molar ratio of 1: 1 with respect to the peptide.
[0062]
Luciferase derived from firefly luciferase (manufactured by Sigma) dissolved in 100 mM sodium carbonate (pH 8.6) to a concentration of 0.1 mg / ml, added to a micro test well, 0.1 ml, and left at 4 ° C. for 12 hours. Was immobilized on the well. The luciferase solution was discarded, and 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 instead, followed by blocking at room temperature for 1 hour. The blocking buffer was discarded, and instead, 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.
[0063]
The FITC-labeled peptide was added at various concentrations to the micro test well on which the luciferase was immobilized. After standing at room temperature for 1 hour, the amount of labeled peptide adsorbed on the well by binding with luciferase was quantified from the fluorescence spectrum of FITC.
[0064]
When the ratio of the amount of labeled peptide adsorbed to the amount of labeled peptide adsorbed is plotted (Scatchard plot), a linear relationship is obtained, and the binding constant 10 ^Ten [l / mol] could be obtained.
[0065]
Similarly, the remaining seven peptides (II) to (VIII) were measured for binding constants using microtest wells labeled with FITC and immobilized with luciferase. A degree of coupling constant was obtained. The coupling constants are also shown below.
[0066]
[Table 1]

[0067]
(Example 2)
It was verified that luciferase can be detected with high quantitativeness using eight kinds of peptides each having the synthesized amino acid sequences (I) to (VIII).
[0068]
Preparation of fluorescently 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 in an 8-fold molar ratio to luciferase, and allowed to react at room temperature for 40 minutes. The labeling reaction was terminated by quickly loading the reaction solution onto a Sephadex G-25 (previously equilibrated with 50 mM phosphate buffer (pH 7.5)) column. The FITC-labeled elution fraction was collected, applied to a CM-52 column (previously equilibrated with 10 mM phosphate buffer (pH 7.5)), and 10 to 150 mM sodium phosphate buffer (pH 7.5). Were eluted with a linear gradient, and various FITC-labeled luciferase fractions were fractionated. For each fraction, the amount of luciferase was quantified from the absorbance at 280 nm, and the amount of FITC introduced from the absorbance at 494 nm was quantified. A FITC-labeled luciferase fraction was prepared at a molar ratio of 1: 1 with respect to luciferase.
[0069]
Measurement of luciferase by competitive method
Synthetic peptide (I) is dissolved in 100 mM sodium carbonate (pH 8.6) to 100 pmol / ml, 0.1 ml is added to a micro test well, and the peptide (I) is left at 4 ° C. for 12 hours. Immobilized on wells. Discard the peptide (I) solution and add 5 mg / ml bovine serum albumin (Sigma), 100 mM sodium carbonate (pH 8.6) containing 0.02% sodium azide (blocking buffer) for 1 hour at room temperature. Blocked. The blocking buffer was discarded, and instead, 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.
[0070]
A fixed amount of 1 pmol of the FITC-labeled luciferase and 0.1 to 10 pmol of unlabeled luciferase were added to micro test wells on which peptide (I) was immobilized. After standing at room temperature for 1 hour, the luciferase solution was taken out, the wells were thoroughly washed with TBST buffer, and the labeled luciferase adsorbed on the wells by binding with peptide (I) was quantified from the fluorescence amount of FITC.
[0071]
A calibration curve was created by plotting the amount of labeled luciferase adsorbed against the amount of unlabeled luciferase input. FIG. 1 shows an example of the created calibration curve. The fluorescence intensity on the vertical axis in the plot of Fig. 1 indicates the fluorescence intensity observed when 1 pmol of labeled luciferase is added and bound to 10 pmol of peptide (I) without adding unlabeled luciferase. 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.
[0072]
Similarly, with respect to the remaining seven peptides (II) to (VIII), each peptide was immobilized on a micro test well, and a quantitative test was conducted by the competitive method of unlabeled luciferase using FITC-labeled luciferase. As a result, 0.1 to 10 pmol of luciferase contained in the sample could be quantified for any of peptides (II) to (VIII).
[0073]
【The invention's effect】
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 that exhibits specific binding to luciferase. Such a peptide is obtained by adding a labeling substance to the peptide chain, In addition, it can be easily carried and fixed on an appropriate support. Utilizing these characteristics, for example, when the sample is brought into contact with a sample containing luciferase, the luciferase in the sample can be captured in the vicinity of a predetermined support through binding to such a peptide. Therefore, by utilizing a competitive reaction with a known concentration of luciferase provided with a labeling substance, it is possible to detect and further quantify luciferase in a biological sample in vitro.
[0074]
[Sequence Listing]

[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is an example of a quantitative curve used for quantification of luciferase by a competition method using binding / fixation by a luciferase-binding peptide of the present invention.

Claims (2)

The following seven types of amino acid sequence (I) ~ (IV), (VI) ~ composed of one kind of amino acid sequence selected from the group consisting of (VIII), peptides with binding to luciferase.
Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Leu-Ala-Arg-Trp (I) (SEQ ID NO: 1)
Trp-His-Asn-Trp-Thr-Trp-Thr-Gly-Leu-Pro-Gly-Gln (II) (SEQ ID NO: 2)
Gly-His-Trp-Thr-Ala-Ser-Thr-Asn-Tyr-Val-Arg-Pro (III) (SEQ ID NO: 3)
Gln-Thr-Thr-His-Trp-Asp-Ser-Ala-Lys-Tyr-Pro-Ala (IV) (SEQ ID NO: 4)
Phe-His-Trp-Trp-Tyr-Thr-Ile (VI) (SEQ ID NO: 6)
Phe-Pro-Ala-His-Ala-Ser-Arg (VII) (SEQ ID NO: 7)
Tyr-Ser-Phe-Glu-Arg-Ser-Lys (VIII) (SEQ ID NO: 8) As a means for detecting luciferase,
The following seven types of amino acid sequence (I) ~ (IV), which comprises a peptide capable of binding to one type of the amino acid sequence, a luciferase selected from the group consisting of (VI) ~ (VIII) Luciferase detection kit.
Lys-His-Val-Asn-Glu-Leu-Arg-Glu-Leu-Ala-Arg-Trp (I) (SEQ ID NO: 1)
Trp-His-Asn-Trp-Thr-Trp-Thr-Gly-Leu-Pro-Gly-Gln (II) (SEQ ID NO: 2)
Gly-His-Trp-Thr-Ala-Ser-Thr-Asn-Tyr-Val-Arg-Pro (III) (SEQ ID NO: 3)
Gln-Thr-Thr-His-Trp-Asp-Ser-Ala-Lys-Tyr-Pro-Ala (IV) (SEQ ID NO: 4)
Phe-His-Trp-Trp-Tyr-Thr-Ile (VI) (SEQ ID NO: 6)
Phe-Pro-Ala-His-Ala-Ser-Arg (VII) (SEQ ID NO: 7)
Tyr-Ser-Phe-Glu-Arg-Ser-Lys (VIII) (SEQ ID NO: 8)

Images