CN114957488A - Method for stably and efficiently expressing aromatic hydrocarbon receptor protein and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and relates to a production method and application of a recombinant protein, in particular to a method for stably and efficiently expressing an aryl hydrocarbon receptor protein and its application. In the expression of aryl hydrocarbon receptor protein (AhR), a molecular chaperone is used to assist the co-expression of the AhR protein, so that the stable and high expression of AhR can be achieved. The AhR protein prepared by the method has uniform properties, good stability, and has the activity of binding small molecules of pollutants, and can be used to develop various equipment for environmental pollutant detection and the like.

Description

A method for stable and efficient expression of aryl hydrocarbon receptor protein and its application

technical field

The invention belongs to the technical field of genetic engineering, and relates to a production method and application of a recombinant protein, in particular to a method for stably and efficiently expressing an aryl hydrocarbon receptor protein and its application.

Background technique

Dioxin-like chemicals are a class of substances with extensive toxic effects and difficult to degrade, and they exist widely in the environment, causing great harm to the human body. One of the most typical dioxin-like pollutants is 2,3,7,8 tetrachlorodibenzodioxin (TCDD). The high-sensitivity detection of dioxins is of great significance to my country's environmental protection and human health protection, especially under the trend of increasingly frequent international trade, it can reduce the harm of dioxins carried by foreign goods. Traditional dioxin detection methods are mainly chromatography and mass spectrometry, but there are obvious defects, such as high detection cost, low precision, and high difficulty. Biochip detection methods gradually developed in recent years can make up for these shortcomings, but the core components in biochips—protein molecules that specifically bind to dioxins—are difficult to obtain. The best candidate protein is the aryl hydrocarbon receptor (AhR) protein, which is a protein that naturally binds dioxins in organisms. Therefore, it is of great significance to study a process method that can stably prepare aromatic hydrocarbon receptors for the detection of environmental pollutants in my country.

AhR protein belongs to the PER-ARNT-SIM (PAS) family of proteins and is a class of transcription factors that mediate the response of cells to many cellular metabolites and environmental pollutants. It is closely related to cytotoxicity, vascular development, and immune development. It is the only responsive molecule of dioxin in the organism. In the cytoplasm of cells, AhR binds to molecular chaperones to form a resting-state complex. When substrate molecules (such as dioxin, etc.) enter the cell, AhR can specifically bind to the substrate, expose the nuclear localization signal, and then transfer to the nucleus to form an activated complex with the nuclear transport protein ARNT, thereby regulating the downstream response gene expression. AhR has been widely studied as a star molecule for a long time, but most studies have found that the receptor domain (PAS-B) of AhR is difficult to obtain stable protein products through recombinant expression and purification, and the protein atom structure containing this region has also been not parsed. Recently, some research groups have obtained full-length AhR protein or AhR truncated protein containing substrate-binding region by prokaryotic expression (S.Uemura et al., 2020, J.Biochem; A.Stinn et al., 2021, Biosensors). However, these studies either prepared a small amount of AhR protein by special host cells, or prepared the activated complex of AhR binding ARNT protein, but they did not prepare a large amount of expressed AhR resting state protein in a stable state, which cannot be used in for biochip applications.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for stably and efficiently expressing an aryl hydrocarbon receptor protein and its application.

To achieve the above object, the present invention adopts the technical scheme as follows:

A method for stable and high-efficiency expression of aryl hydrocarbon receptor protein. In the expression of aryl hydrocarbon receptor protein (AhR), molecular chaperones are used to assist the co-expression of AhR protein, so that the stable and high-efficiency expression of AhR can be achieved; wherein, the aryl hydrocarbon receptor protein (AhR ) and molecular chaperones in a mass ratio of 1:1.

The AhR protein is an amino acid sequence comprising a PAS-B domain.

The AhR protein is human AhR or murine AhR.

The molecular chaperone is one or more of human or murine Hsp90, XAP2 and p23.

Sequence selection of the AhR protein described above. AhR proteins from different species have similar domain composition, including bHLH domain, PAS-A domain, PAS-B domain and TAD transcriptional activation domain from N-terminal to C-terminal, respectively. Among them, the PAS-B domain is a functional region that binds to small molecules of pollutants, so the recombinantly expressed protein product must contain the PAS-B domain. Other functional areas can be adjusted according to different purposes. For example, truncations of bHLH/PAS-A/PAS-B (see Example 1), truncations of PAS-A/PAS-B, truncations of PAS-B/TAD, and the like. AhR protein molecules of different species have similar functions and high sequence homology. AhR protein sequences from different species can be selected according to specific usage scenarios and usage requirements, such as human AhR (seq ID No.1), Murine AhR (seq ID No. 2, see Example 1), bovine AhR, and the like.

At the same time, molecular chaperone proteins are selected. AhR proteins in organisms can combine with a variety of molecular chaperones to form stable resting-state complex structures, including Hsp90, XAP2 and p23. Among them, the applicant found that Hsp90 protein has the most important auxiliary effect on the expression efficiency and protein stability of AhR. Hsp90 proteins from different species have similar sequences and structures, and Hsp90 proteins from different species can be selected according to specific usage scenarios and usage requirements, such as human Hsp90, mouse Hsp90 (seq ID No. 3, see Example 1) , bovine source Hsp90 and so on.

Further, the aryl hydrocarbon receptor AhR protein is the amino acid sequence shown in any one of SEQ ID No.1 and SEQ ID No.2, or any one of the aforementioned amino acid sequences is substituted, added, or deleted 1 or more. The amino acid sequence of amino acid residues, or a sequence with more than 80% sequence identity to the amino acid sequence shown in any one of SEQ ID No.1 and SEQ ID No.2; or SEQ ID No.1, SEQ ID No.2 Further optimized amino acid sequences, respectively, wherein the AhR protein has the activity of binding contaminant small molecules.

The molecular chaperone can select different kinds of molecular chaperone proteins from different species. Preferably, the molecular chaperones include Hsp90 (SEQ ID No. 3), XPA2 (SEQ ID No. 4), P23 (SEQ ID No. 5), etc., including SEQ ID No. 3, SEQ ID No. 4 and SEQ ID The amino acid sequence shown in any one of No. 5, or the amino acid sequence in which one or more amino acids are substituted, added, or deleted from any of the aforementioned amino acid sequences, or the same as SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 3 The amino acid sequence shown in any of ID No. 5 has a sequence identity of more than 80%, or the amino acid sequence of SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5 are further optimized, respectively.

The aryl hydrocarbon receptor protein further preferably comprises the nucleotide sequence shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, or SEQ ID No.10.

A method for stable and high-efficiency expression of aryl hydrocarbon receptor protein, wherein the molecular chaperone and the AhR protein are co-constructed on the same vector for co-expression; or, the molecular chaperone and the AhR protein are respectively constructed on the same vector, and then co-transfected into host cells Perform co-expression; or, add purified molecular chaperone protein and AhR protein to the expression system for co-expression.

A stable and highly expressed aryl hydrocarbon receptor protein is obtained according to the described method.

An application of the aryl hydrocarbon receptor protein stably and efficiently expressed by the method, and the above-mentioned method obtains the application of the aryl hydrocarbon receptor protein stably and highly expressed in the detection of pollutants.

An expression vector for detecting pollutant dioxin-like chemical substances, the vector contains the aryl hydrocarbon receptor protein obtained by the above-mentioned method to obtain stable and high-efficiency expression.

The molecular chaperone and AhR protein are separately or jointly constructed on the same vector for co-expression, wherein the vector is pFastBac-Dual vector, pETDuet-1 vector, and pBudCE4.1 vector.

A host cell for detecting pollutant dioxin-like chemicals, the host cell containing the aryl hydrocarbon receptor protein obtained by the above-mentioned method to obtain stable and high expression.

The host cells are insect cells, Escherichia coli or yeast.

The above-mentioned proteins for detecting dioxin-like chemical substances can be prepared into pharmaceutical compositions, medical equipment, biochip products, tissue engineering products, cosmetics or health products, testing supplies, and the like.

Compared with the prior art, the present invention has the following characteristics:

The invention utilizes a specific molecular chaperone Hsp90 to assist the AhR protein to express it stably and efficiently, utilizes the obtained protein to have the activity of specifically binding pollutant small molecules, and is used for the detection of environmental pollutants. The invention has a simple production process and is easy to popularize. Various equipment for the detection of environmental pollutants has a wide range of application prospects; specifically:

(1) The present invention realizes the stable and high-efficiency expression of the receptor binding region of the AhR protein for the first time through the assistance of a preferred molecular chaperone.

(2) The AhR protein complex prepared by the present invention is in a quiescent state, and experiments have confirmed that it can recognize and bind to small molecules of pollutants and has biological activity.

(3) The preparation process of the invention is simple, the protein properties are good, and the invention has wide application prospects in the detection of environmental pollutants and the like.

Description of drawings

Figure 1 is an SDS-PAGE electrophoresis image of the AhR protein and molecular chaperone binding complex purified by the present invention. Hsp90, XAP2 and p23 molecular chaperones are stably combined with AhR and purified together.

Fig. 2 is a Native-Page electrophoresis image of the AhR protein complex purified by the present invention, showing that the molecular chaperone and the AhR protein are stably combined to form a uniform protein complex.

Figure 3 shows that the AhR protein complex purified by the present invention presents a single main peak in size exclusion chromatography detection, showing that the molecular chaperone and AhR protein are stably combined to form a uniform protein complex.

Fig. 4 is the detection result of the peptide fingerprint of the AhR protein complex purified by the present invention. The red color represents the peptide detected by MALDI mass spectrometry, and the sequence is the theoretical sequence of the AhR target protein, which proves that the AhR target protein exists in the protein complex, especially the PAS-B domain.

Figure 5 shows the protein composition in the AhR protein complex purified by the present invention analyzed by Orbitrap mass spectrometry, showing that AhR, Hsp90, XAP2 and P23 and other components exist in the complex.

Fig. 6 is the detection result of analyzing the AhR protein complex obtained by the purification of the present invention to bind the contaminant small molecule TCDD by using microcalorimetry electrophoresis MST. It was shown that the AhR protein in the complex can efficiently bind TCDD small molecules.

Figure 7 is a diagram of cryopreserved cells.

Detailed ways

The following examples are provided to illustrate the invention. It should be understood by those skilled in the art that the embodiments are merely illustrative and not restrictive. The present invention is limited only by the scope of the appended claims.

Further description is also provided below to facilitate understanding of the present invention.

Aiming at the problem that the AhR protein is easy to form inclusion bodies when expressed alone, the present invention can achieve the purpose of soluble and stable expression of the AhR protein by adding a suitable molecular chaperone protein (such as Hsp90) into the expression system. Among them, the AhR protein can be selected from the amino acid sequence of the PAS-B domain derived from different species, and the molecular chaperone can be selected from the amino acid sequence of Hsp90 protein, XAP2 protein and P23 protein derived from different species. The AhR protein prepared by the method has uniform properties, good stability, and has the activity of binding small molecules of pollutants, and can be used to develop various equipment for environmental pollutant detection and the like.

In the present invention, the selected AhR proteins can be derived from various species, because AhR proteins derived from different species all contain the PAS-B domain that binds small pollutant molecules, and can play similar functions. For example, for the sequence of the human AhR protein, refer to the sequence in the Uniprot database ( https://www.uniprot.org/uniprot/P35869 , index number P35869), and its complete protein sequence is as follows:

The bold underlined part in the above sequence is the amino acid sequence (SEQ ID No. 1) selected in the present invention, which includes the PAS-B receptor binding region and is named as hAhR-1 sequence. Its corresponding DNA sequence optimized according to the insect expression system is SEQ ID No.6.

For the sequence of the murine AhR protein, refer to the sequence in the Uniprot database ( https://www.uniprot.org/uniprot/P30561 ; index number P30561 ), and its complete protein sequence is as follows:

The bold underlined part in the above sequence is the amino acid sequence (SEQ ID No. 2) selected in the present invention, which includes the PAS-B receptor binding region, and is named as the mAhR-1 sequence. Its corresponding DNA sequence optimized according to the insect expression system is SEQ ID No.7.

In the present invention, the selected molecular chaperones can be molecular chaperones derived from different species and different species, all of which can help the soluble expression of AhR, including Hsp90, XAP2 and p23. Among them, Hsp90 protein is selected in this application, and Hsp90 proteins of different species have basically the same structure and function, and can have similar effects.

Selected in the following embodiment 1 is the murine AhR protein sequence, with reference to the sequence in the Uniprot database ( https://www.uniprot.org/uniprot/P34058#structure ; index number P34058), its complete protein sequence is as follows:

MPEEVHHGEEEVETFAFQAEIAQLMSLIINTFYSNKEIFLRELISNASDALDKIRYESLTDPSKLDSGKELKIDIIPNPQERTLTLVDTGIGMTKADLINNLGTIAKSGTKAFMEALQAGADISMIGQFGVGFYSAYLVAEKVVVITKHNDDEQYAWESSAGGSFTVRADHGEPIGRGTKVILHLKEDQTEYLEERRVKEVVKKHSQFIGYPITLYLEKEREKEISDDEAEEEKGEKEEEDKEDEEKPKIEDVGSDEEDDSGKDKKKKTKKIKEKYIDQEELNKTKPIWTRNPDDITQEEYGEFYKSLTNDWEDHLAVKHFSVEGQLEFRALLFIPRRAPFDLFENKKKKNNIKLYVRRVFIMDSCDELIPEYLNFIRGVVDSEDLPLNISREMLQQSKILKVIRKNIVKKCLELFSELAEDKENYKKFYEAFSKNLKLGIHEDSTNRRRLSELLRYHTSQSGDEMTSLSEYVSRMKETQKSIYYITGESKEQVANSAFVERVRKRGFEVVYMTEPIDEYCVQQLKEFDGKSLVSVTKEGLELPEDEEEKKKMEESKAKFENLCKLMKEILDKKVEKVTISNRLVSSPCCIVTSTYGWTANMERIMKAQALRDNSTMGYMMAKKHLEINPDHPIVETLRQKAEADKNDKAVKDLVVLLFETALLSSGFSLEDPQTHSNRIYRMIKLGLGIDEDEVTAEEPSAAVPDEIPPLEGDEDASRMEEVD

The above sequence is the amino acid sequence (SEQ ID No. 3) selected in the present invention, named as mHsp90, and its corresponding DNA sequence optimized according to the insect expression system is SEQ ID No. 8.

In addition, XAP2 and P23 from different species also have auxiliary effects on the recombinant expression of AhR. For example, what is selected in embodiment 1 is the XAP2 protein of murine origin, with reference to the sequence in the Uniprot database ( https://www.uniprot.org/uniprot/008915 ; index number 008915 ), its complete protein sequence is as follows:

MADLIARLREDGIQKRVIQEGRGELPDFQDGTKATFHFRTLHSDNEGSVIDDSRTRGKPMELIVGKKFKLPVWETIVCTMREGEIAQFLCDIKHVVLYPLVAKSLRNIAEGKDPLEGQRHCCGIAQMHEHSSLGHADLDALQQNPQPLIFHIEMLKVESPGTYQQDPWAMTDEEKAKAVPVIHQEGNRLYREGQVKEAAAKYYDAIACLKNLQMKEQPGSPDWIQLDLQITPLLLNYCQCKLVAQEYYEVLDHCSSILNKYDDNVKAYFKRGKAHAAVWNAQEAQADFAKVLELDPALAPVVSRELRALETRIRQKDEEDKARFRGIFSH

The above-mentioned bold underlined partial sequence is the amino acid sequence (SEQ ID No. 4) selected by the present invention, named as mXAP2, and its corresponding DNA sequence optimized according to the insect expression system is SEQ ID No. 9.

Also selected in embodiment 1 is the P23 protein of murine origin, with reference to the sequence in the Uniprot database ( https://www.uniprot.org/uniprot/Q9R0Q7 ; index number Q9R0Q7 ), its complete protein sequence is as follows:

MQPASAKWYDRRDYVFIEFCVEDSKDVNVNFEKSKLTFSCLGGSDNFKHLNEIDLFHCIDPNDSKHKRTDRSILCCLRKGESGQSWPRLTKERAKLNWLSVDFNNWKDWEDDSDEDMSNFDRFSEMMDHMGGDEDVDLPEVDGADDDSQDSDDEKMPDLE

The above sequence is the amino acid sequence (SEQ ID No. 5) selected by the present invention, named as mP23, and its corresponding DNA sequence optimized according to the insect expression system is SEQ ID No. 10.

Example 1 Insect cell expression method of AhR protein

1. According to the codon characteristics preferred by the insect expression system, for mAhR-1 (SEQ ID No. 2), mHsp90 (SEQ ID No. 3), mXAP2 (SEQ ID No. 4), mP23 (SEQ ID No. 5) proteins The corresponding gene sequences are optimized and gene synthesis is performed, that is, the optimized sequences are SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, and SEQ ID No.10 in sequence.

2. The pFastBac-Dual vector plasmid was linearized with restriction endonuclease XhoI, digested at 37°C for 3 hours, then added alkaline phosphatase and incubated at 37°C for 0.5 hours, and purified by DNA gel recovery kit.

3. Use primers SEQ ID No.11 and SEQ ID No.12 to PCR amplify the DNA sequence fragment (SEQ ID No.7) of mAhR-1. The PCR enzyme used in the amplification is NEB product Q5 polymerase, and the amplification conditions are ①98°C , 2 minutes; ② 98°C, 10 seconds; ③ 72°C for 45 seconds (steps ②, ③ cycle 30 times); ④ 72°C for 5 minutes. Make sure that the two ends of the target sequence and the two ends of the linearized vector have overlapping regions of 26-30 bases, and directly synthesize a Strep tag at the N-terminus of the sequence for protein purification. Mix the linearized vector with the DNA sequence fragment of mAhR-1 in a molar ratio of 1:1.2, add NEBuilder DNA self-assembly mixture reagent, incubate at 50°C for 1 hour, and incubate at 70°C for 10 minutes, and then transform the self-assembled DNA product. to DH5α competence.

4. Pick the transformed single colony, identify the positive clones, and extract the pFastBac-Dual-mAhR plasmid.

5. On the basis of making the pFastBac-Dual-mAhR plasmid, the vector was linearized with restriction endonuclease NotI, and the DNA sequence fragment of mHsp90 was PCR amplified with primers SEQ ID No. 13 and SEQ ID No. 14 (SEQ ID No. 14). ID No.8), the PCR enzyme used in the amplification was NEB product Q5 polymerase, and the amplification conditions were ①98°C, 2 minutes; ②98°C, 10 seconds; ③72°C, 75 seconds (steps ②, ③ cycle 30 times); ④72 °C for 5 minutes. Subsequently, the pFastBac-Dual-mAhR plasmid and mHsp90 DNA fragment were assembled with DNA self-assembly mixture reagent, and transformed into DH5α competent. The transformed single colony was picked, and the positive clones were identified, and the plasmid pFastBac-Dual-mAhR-mHsp90 was obtained by extraction.

6. The DNA sequences of mXAP2 (primers SEQ ID No. 15 and SEQ ID No. 16), mP23 (primers SEQ ID No. 17 and SEQ ID No. 18) proteins were constructed into another pFastBac-Dual vector plasmid by a similar method The PCR enzyme used in the amplification was NEB product Q5 polymerase, and the amplification conditions were ① 98°C, 2 minutes; ② 98°C, 10 seconds; ③ 72°C for 60 seconds (steps ②, ③ cycle 30 times); The other experimental procedures for constructing plasmids are the same as in steps 3, 4, and 5 to obtain the pFastBac-Dual-mXAP2-mP23 plasmid.

7. The pFastBac-Dual-mAhR-mHsp90 and pFastBac-Dual-mXAP2-mP23 plasmids prepared above were transformed into DH10Bac competent cells respectively, and positive clones were obtained through blue-white screening, and bacmids were extracted.

8. Take 2 milliliters of healthy insect cells (this example adopts Spodoptera frugiperda sf9 cells) cultured in the insect cell culture medium (Ishengke IB905 medium; article number L11001; the medium described later is this medium). ), added to a 35 mm plastic dish, and allowed to stand for 2 hours to allow the cells to adhere. After 2 hours, the medium in the dish was aspirated and 1 ml of the insect cell culture medium described above was added. Take 3 micrograms of bacmid of two different constructions and mix with 8 microliters of transfection reagent (GibcoCellfectin reagent, Cat. No. 10362100) for 20 minutes, and then add the mixture to confluent insect cells (Spodoptera frugiperda sf9 in this example). 1 ml of the above-described insect cell culture medium supplemented with streptomycin and penicillin was added to the plate overnight.

9. Collect the supernatant after 6-7 days, which is the P1 virus concentrate. 100 μl was added to 200 ml of healthy insect cells (Spodoptera frugiperda sf9 cells were used in this example), and after 4-5 days, the cell death rate reached 50%, and the supernatant was collected, which was the P2 virus concentrate.

10. Take 10 ml of each of the P2 virus concentrates prepared from the two bacmids and add them to 600 ml of healthy insect cells for mass expression. After 48 hours, centrifuge at 2000 g to collect the cells, which can be used immediately or stored in a negative 80°C refrigerator. (See Figure 7).

Example 2 Purification of AhR protein

1. Dilute the AhR protein-expressing insect cells obtained in Example 1 above with a buffer (30 mM HEPES, pH 7.5, 20 mM NaCl, 10 mM MgCl ₂ ), and stir well. Then, the cells were disrupted with a sonicator, and the parameters were set to 8 seconds intermittently, 3 seconds ultrasound, a total time of 15 minutes, a power of 300 watts, and two rounds of ultrasound.

2. After ultrasonication, centrifuge the broken liquid with a high-speed centrifuge at 16,000 rpm for 30 minutes, discard the precipitate, and leave the supernatant. The gravity column with Strep medium was washed twice with buffer and cooled in a 4°C refrigerator.

3. After centrifugation, the supernatant of insect cells assisted by different molecular chaperones expressing AhR protein was added to a gravity column with a diameter of 1 cm and a length of 20 cm containing Strep medium for natural flow-through, and the flow-through liquid was added to the gravity column again. , repeat 3-4 times to fully bind the mAhR to the Strep medium. Then buffer (30 mM HEPES, pH 7.5, 20 mM NaCl, 10 mM MgCl ₂ ) was added to the gravity column to wash thoroughly until no impurities were eluted. Finally, an elution buffer (30 mM HEPES, pH 7.5, 20 mM NaCl, 10 mM MgCl ₂ , 5 mM d-Desthiobiotin) was added to elute the protein until no target protein was eluted.

4. Concentrate the eluted protein to 300 microliters and pass through molecular sieves (such as Superdex ^TM 200 10/300GL), and the peak component of about 12 ml of molecular sieves is the mAhR complex (see Figure 3).

5. Concentrate the components at the peak positions of molecular sieves and perform SDS-PAGE electrophoresis (see Figure 1) and Native-PAGE electrophoresis (see Figure 2). The lower components are uniform.

Example 3 Identification of AhR protein and intact complex

1) The band corresponding to about 50KD on the SDS-PAGE gel was cut out, and after DTT reduction and iodoacetamide alkylation treatment, trypsin was added for enzymatic digestion overnight. The peptides obtained after enzymatic hydrolysis were desalted with C18ZipTip, and then mixed with the matrix α-cyano-4-hydroxycinnamic acid (CHCA) to spot plate. Finally, the analysis was performed with a matrix-assisted laser desorption ionization-time-of-flight mass spectrometer MALDI-TOF/TOF UltraflextremeTM, Brucker, Germany.

2) By conducting a database search from the Peptide Mass Fingerprint page on the www.matrixscience.com website. Protein identification results were obtained based on primary mass spectrometry of peptides generated after enzymatic digestion. Retrieval parameters: Trypsin digestion, set two missed cleavage sites. Alkylation of cysteine was set as a fixed modification and oxidation of methionine as a variable modification. The database used for identification was Swissprot NCBIProt (see Figure 4).

3) The band of the protein complex on the Native-PAGE gel was excised, and after decolorization, DTT reduction and iodoacetamide alkylation treatment, trypsin was added for enzymatic digestion overnight, and then the peptide was extracted with 60% acetonitrile. The peptide mixture obtained after enzymatic hydrolysis was analyzed by liquid chromatography-linear ion trap-orbitrap (nanoLC-LTQ-Orbitrap XL, Thermo, San Jose, CA) mass spectrometer. The chromatographic analysis column is a laboratory-filled C18 reversed-phase column, with an inner diameter of 75 microns and a length of 15 cm, and is filled with 3 microns ReproSil-Pur C18-AQ ((Dr. Maisch GmbH, Ammerbuch) packing, and the loading column is 150 microns in inner diameter. 3 cm long, filled with 5u ReproSil-Pur C18-AQ ((Dr. Maisch GmbH, Ammerbuch) packing material. The mobile phase was A: 0.1% FA/H2O, B: 0.1% FA/80% ACN, and the flow rate was 300 nanoliters Every minute. A 90 minute gradient was used.

4) Data analysis Proteome Discoverer (version 1.4.0.288, Thermo Fischer Scientific) software was used for analysis. MS2 spectra were searched against the Uniprot_proteome_mouse_2019 database using the SEQUEST search engine. Retrieval parameters: Trypsin enzymatic hydrolysis, two missed cleavage sites, the mass error of precursor ions is less than 20ppm, and the mass error of fragment ions is less than 0.6Da. The alkylation of cysteine was set as a fixed modification and the oxidation of methionine as a variable modification. The retrieved peptides and spectral matches (PSM) were filtered using the Percolator algorithm with a q value of less than 1% (1% FDR). The retrieved peptides were combined into proteins using strict maximum parsimony principles (see Figure 5).

Furthermore, it can be seen from Fig. 5 that this technique can successfully purify the complex of AhR and molecular chaperone.

Example 4 Activity verification of AhR protein

1. Use MonolithTM RED-NHS second-generation protein labeling kit (nanotemper company kit; product number: MO-L011) to label the obtained protein complex.

2. Prepare 16 EP tubes and label No. 1-16 in sequence. Take 5 microliters of buffer (30mM HEPES, PH7.5, 20mMNaCl, 10mM MgCl ₂ ) into tubes 2-16, take 10 microliters of 10 micromolar TCDD small molecules into tube number 1, from tube number 1 Add 5 microliters to tube No. 2 and mix well, take 5 microliters from tube No. 2 and add it to tube No. 3 and mix well, operate in sequence, and finally take 5 microliters from tube No. 16 and discard.

3. Add 5 microliters of 1 micromolar labeled protein complex to each EP tube, mix well, and load the sample with a capillary. The kd values were determined using the Binding Affinity mode of the Monolith™ NT.115 instrument (see Figure 6).

It can be seen from FIG. 6 that the complex of AhR and molecular chaperone purified by this technology has the activity of binding the substrate small molecule TCDD.

sequence

SEQ ID NO. 1 (hAhR-1)

VKPIPAEGIKSNPSKRHRDRLNTELDRLASLLPFPQDVINKLDKLSVLRLSVSYLRAKSFFDVALKSSPTERNGGQDNCRAANFREGLNLQEGEFLLQALNGFVLVVTTDALVFYASSTIQDYLGFQQSDVIHQSVYELIHTEDRAEFQRQLHWALNPSQCTESGQGIEEATGLPQTVVCYNPDQIPPENSPLMERCFICRLRCLLDNSSGFLAMNFQGKLKYLHGQKKKGKDGSILPPQLALFAIATPLQPPSILEIRTKNFIFRTKHKLDFTPIGCDAKGRIVLGYTEAELCTRGSGYQFIHAADMLYCAESHIRMIKTGESGMIVFRLLTKNNRWTWVQSNARLLYKNGRPDYIIVTQRPLTDEEGTEHLRKRNTKLPFMFTTGEAVLYEATNPFPAIMDPLPLRTKNGTSGKDSATTSTLSKDSLNPSSLLAAMMQQDESIYLYPASST

SEQ ID NO. 2 (mAhR-1)

MSSGANITYASRKRRKPVQKTVKPIPAEGIKSNPSKRHRDRLNTELDRLASLLPFPQDVINKLDKLSVLRLSVSYLRAKSFFDVALKSTPADRNGGQDQCRAQIRDWQDLQEGEFLLQALNGFVLVVTADALVFYASSTIQDYLGFQQSDVIHQSVYELIHTEDRAEFQRQLHWALNPDSAQGVDEAHGPPQAAVYYTPDQLPPENASFMERCFRCRLRCLLDNSSGFLAMNFQGRLKYLHGQNKKGKDGALLPPQLALFAIATPLQPPSILEIRTKNFIFRTKHKLDFTPIGCDAKGQLILGYTEVELCTRGSGYQFIHAADMLHCAESHIRMIKTGESGMTVFRLFAKHSRWRWVQSNARLIYRNGRPDYIIATQRPLTDEEGREHLQKRSTSLPF

SEQ ID NO. 3 (mHsp90)

MPEEVHHGEEEVETFAFQAEIAQLMSLIINTFYSNKEIFLRELISNASDALDKIRYESLTDPSKLDSGKELKIDIIPNPQERTLTLVDTGIGMTKADLINNLGTIAKSGTKAFMEALQAGADISMIGQFGVGFYSAYLVAEKVVVITKHNDDEQYAWESSAGGSFTVRADHGEPIGRGTKVILHLKEDQTEYLEERRVKEVVKKHSQFIGYPITLYLEKEREKEISDDEAEEEKGEKEEEDKEDEEKPKIEDVGSDEEDDSGKDKKKKTKKIKEKYIDQEELNKTKPIWTRNPDDITQEEYGEFYKSLTNDWEDHLAVKHFSVEGQLEFRALLFIPRRAPFDLFENKKKKNNIKLYVRRVFIMDSCDELIPEYLNFIRGVVDSEDLPLNISREMLQQSKILKVIRKNIVKKCLELFSELAEDKENYKKFYEAFSKNLKLGIHEDSTNRRRLSELLRYHTSQSGDEMTSLSEYVSRMKETQKSIYYITGESKEQVANSAFVERVRKRGFEVVYMTEPIDEYCVQQLKEFDGKSLVSVTKEGLELPEDEEEKKKMEESKAKFENLCKLMKEILDKKVEKVTISNRLVSSPCCIVTSTYGWTANMERIMKAQALRDNSTMGYMMAKKHLEINPDHPIVETLRQKAEADKNDKAVKDLVVLLFETALLSSGFSLEDPQTHSNRIYRMIKLGLGIDEDEVTAEEPSAAVPDEIPPLEGDEDASRMEEVD

SEQ ID NO. 4 (mXAP2)

MADLIARLREDGIQKRVIQEGRGELPDFQDGTKATFHFRTLHSDNEGSVIDDSRTRGKPMELIVGKKFKLPVWETIVCTMREGEIAQFLCDIKHVVLYPLVAKSLRNIAEGKDPLEGQRHCCGIAQMHEHSSLGHADLDALQQNPQPLIFHIEMLKVESPGTYQQDPWAMTDEEKAKAVPVIHQEGNRLYREGQVKEAAAKYYDAIACLKNLQMKEQPGSPDWIQLDLQITPLLLNYCQCKLVAQEYYEVLDHCSSILNKYDDNVKAYFKRGKAHAAVWNAQEAQADFAKVLELDPALAPVVSRELRALETRIRQKDEEDKARFRGIFSH

SEQ ID NO. 5 (mP23)

MQPASAKWYDRRDYVFIEFCVEDSKDVNVNFEKSKLTFSCLGGSDNFKHLNEIDLFHCIDPNDSKHKRTDRSILCCLRKGESGQSWPRLTKERAKLNWLSVDFNNWKDWEDDSDEDMSNFDRFSEMMDHMGGDEDVDLPEVDGADDDSQDSDDEKMPDLE

SEQ ID No. 6 (hAhR-1-DNA)

GTAAAACCTATTCCGGCAGAAGGCATTAAAAGCAACCCGAGCAAAAGACATCGTGACCGGTTAAATACAGAGCTGGATCGTCTGGCAAGCCTTTTACCGTTCCCGCAGGATGTTATTAATAAACTGGACAAACTGAGTGTACTGCGCCTGAGCGTTAGCTATTTACGTGCTAAAAGTTTTTTCGACGTGGCTCTGAAATCTAGTCCGACCGAGCGTAATGGTGGGCAGGATAACTGCCGGGCAGCAAACTTTCGCGAAGGTCTGAATTTACAGGAGGGGGAATTTCTTCTGCAGGCTCTGAATGGTTTTGTTCTGGTTGTTACAACCGATGCACTGGTTTTTTATGCAAGCTCTACAATTCAGGATTATTTAGGGTTTCAACAGAGCGATGTGATACATCAGTCTGTTTATGAATTAATCCACACCGAGGACCGTGCAGAGTTTCAAAGACAACTGCATTGGGCACTGAACCCAAGCCAGTGTACCGAGAGCGGGCAAGGTATCGAGGAAGCAACCGGTCTTCCACAGACCGTGGTTTGTTATAATCCGGATCAGATTCCGCCAGAAAATAGCCCCCTGATGGAACGCTGTTTTATTTGTCGCCTGAGATGTTTACTGGACAATAGCTCAGGCTTTCTGGCAATGAACTTTCAGGGTAAACTGAAATATCTTCACGGTCAGAAGAAAAAGGGTAAAGATGGAAGTATTCTGCCGCCGCAACTTGCACTGTTTGCAATTGCAACCCCCCTGCAGCCTCCGTCTATCCTGGAAATTCGCACCAAAAATTTTATCTTTCGTACCAAACACAAGCTGGATTTTACCCCCATTGGTTGTGATGCCAAAGGTCGTATTGTTTTAGGCTATACCGAAGCAGAACTGTGCACCCGGGGTTCAGGCTATCAGTTTATTCATGCAGCAGATATGCTGTATTGTGCCGAAAGCCACATTCGGATGATTAAGACAGGTGAAAGTGGGATGATTGTTTTTCGTCTGCTTACCA AAAATAACCGTTGGACCTGGGTGCAGAGCAATGCACGTTTATTATATAAGAATGGTCGTCCGGACTATATTATTGTTACACAGCGGCCGTTAACCGACGAGGAGGGTACCGAACATCTGCGTAAACGCAACACCAAATTACCATTTATGTTTACCACCGGTGAGGCGGTGCTGTATGAAGCAACCAACCCGTTTCCGGCAATTATGGACCCTTTACCGCTGCGTACCAAAAACGGTACCTCAGGTAAAGACAGCGCGACCACCAGCACCTTAAGCAAAGATAGCCTGAATCCCAGCAGTCTGCTGGCAGCTATGATGCAGCAGGATGAAAGCATTTATCTGTATCCAGCAAGCAGCACC

SEQ ID No. 7 (mAhR-1-DNA)

ATGAGCAGTGGAGCTAACATTACTTACGCCTCCAGGAAGAGAAGGAAGCCTGTTCAGAAGACTGTGAAGCCAATCCCTGCCGAGGGTATTAAATCCAACCCCTCCAAGCGCCACAGGGACAGATTGAACACCGAGCTGGACCGCCTGGCTAGTTTGTTGCCATTCCCCCAGGACGTGATCAACAAGCTCGACAAACTGTCCGTGCTGCGCCTGTCTGTCTCCTACTTGCGTGCAAAGTCCTTCTTCGACGTGGCTTTGAAATCCACCCCCGCTGACCGTAACGGTGGTCAAGATCAATGCCGCGCTCAGATCCGCGACTGGCAAGATTTGCAGGAGGGTGAGTTCCTCCTGCAGGCTTTGAACGGATTCGTGTTGGTGGTCACCGCTGACGCTCTGGTTTTCTACGCTTCCTCCACCATTCAGGACTACCTGGGATTCCAACAGAGCGACGTGATTCATCAGTCCGTTTACGAGCTGATCCACACCGAGGACCGCGCTGAATTTCAGCGTCAATTGCACTGGGCTCTGAACCCTGACAGTGCTCAGGGTGTGGACGAGGCTCATGGTCCTCCTCAAGCAGCTGTGTACTACACCCCCGACCAGTTGCCTCCAGAAAACGCTTCCTTCATGGAGCGCTGTTTCCGCTGCCGTCTGAGATGTCTCCTGGATAACTCCTCCGGTTTCCTCGCTATGAACTTCCAGGGTCGCCTGAAGTACCTCCACGGTCAAAACAAAAAGGGTAAGGACGGAGCTTTGCTCCCTCCCCAACTGGCTTTGTTCGCTATTGCCACCCCTCTGCAGCCTCCTTCTATCTTGGAAATTCGCACCAAGAACTTCATCTTCCGCACCAAGCACAAGCTCGACTTCACCCCTATCGGTTGCGACGCTAAGGGTCAGCTGATCCTGGGATACACCGAAGTCGAGCTGTGCACCCGTGGTTCTGGATACCAATTCATCCACGCTGCCGACATGCTGCACTGCGCTGAATCTCACATCCGTA TGATCAAGACTGGTGAGTCCGGTATGACCGTGTTCCGCTTGTTCGCTAAGCATTCTCGCTGGCGCTGGGTCCAATCAAACGCTAGACTGATCTACCGTAACGGTCGCCCCGACTACATCATCGCAACCCAAAGACCTCTGACCGACGAGGAGGGTAGGGAACATCTGCAGAAGAGGAGCACCTCCCTGCCTTTC

SEQ ID No. 8 (mHsp90-DNA)

ATGCCTGAGGAGGTGCACCACGGTGAAGAAGAAGTGGAAACCTTCGCTTTCCAGGCTGAGATTGCTCAACTGATGTCCCTCATCATCAACACCTTCTACAGCAACAAGGAGATCTTCCTGAGAGAGCTGATCAGCAACGCTTCCGACGCTCTGGACAAAATCCGTTACGAGTCCCTGACCGATCCTTCCAAACTGGACTCCGGCAAAGAACTGAAGATCGACATCATCCCTAACCCTCAGGAGCGCACCCTCACATTGGTGGATACCGGTATCGGCATGACAAAGGCAGACCTCATCAACAACCTGGGCACCATTGCTAAAAGCGGAACCAAAGCCTTCATGGAGGCTCTGCAAGCAGGTGCTGACATCTCCATGATTGGTCAATTCGGCGTGGGTTTCTACTCCGCTTACCTCGTGGCCGAGAAGGTGGTTGTGATCACAAAGCACAACGACGACGAGCAGTACGCTTGGGAGAGCAGTGCTGGTGGTTCCTTCACTGTCAGGGCTGATCACGGTGAGCCTATCGGTCGTGGTACCAAAGTGATCCTGCATCTGAAGGAGGACCAAACCGAGTACCTCGAAGAGCGCAGGGTGAAGGAGGTGGTTAAGAAGCACAGCCAGTTCATCGGTTACCCCATCACACTGTACTTGGAGAAAGAGCGCGAAAAAGAGATCAGCGACGACGAAGCAGAGGAAGAAAAGGGTGAGAAGGAAGAGGAAGATAAGGAGGACGAGGAGAAGCCAAAGATCGAGGACGTGGGAAGCGACGAGGAGGATGATAGCGGAAAGGACAAGAAGAAGAAGACAAAGAAGATCAAGGAGAAGTACATCGACCAGGAGGAGCTGAACAAGACAAAGCCAATCTGGACAAGGAACCCTGACGACATCACACAGGAGGAGTACGGAGAGTTCTACAAGAGCCTCACAAACGACTGGGAGGACCACCTGGCTGTGAAGCATTTCAGCGTGGAGGGACAGCTGGAGTTCAGGGCTTTGCTGT TCATCCCAAGGAGAGCCCCTTTCGACCTGTTCGAGAACAAAAAGAAGAAGAACAACATCAAACTGTACGTGCGCCGCGTCTTCATTATGGACTCCTGCGACGAGCTGATCCCTGAGTACCTGAACTTCATCCGTGGTGTGGTGGACTCCGAAGACCTGCCTTTGAACATCTCCCGCGAGATGCTGCAGCAGTCCAAGATCCTGAAGGTGATCAGGAAGAACATCGTGAAGAAATGCCTGGAGTTGTTCTCCGAGTTGGCTGAAGACAAAGAGAACTACAAGAAGTTCTACGAGGCATTCTCCAAGAACCTGAAGCTGGGTATCCATGAGGACTCCACCAACCGCCGTCGTTTGTCTGAGCTGCTGCGTTACCACACCAGCCAGTCTGGTGACGAGATGACCTCCCTGTCCGAATACGTGTCCCGCATGAAGGAAACTCAGAAATCAATTTACTACATCACTGGTGAATCCAAGGAGCAAGTGGCTAACTCCGCTTTCGTCGAGCGTGTGCGTAAGAGAGGTTTCGAGGTGGTGTACATGACCGAGCCCATCGACGAATACTGCGTCCAGCAGCTGAAGGAGTTCGACGGTAAGTCCTTGGTGAGCGTCACCAAGGAGGGTCTGGAGTTGCCAGAGGATGAGGAGGAGAAGAAGAAGATGGAAGAGAGCAAGGCAAAGTTCGAGAACCTCTGCAAGTTGATGAAGGAGATCCTGGACAAGAAGGTGGAGAAGGTGACTATCAGCAACAGGCTGGTGAGCAGTCCATGCTGCATCGTGACCAGCACCTACGGATGGACCGCAAACATGGAGAGGATCATGAAGGCACAAGCCCTGCGCGACAACAGCACAATGGGATACATGATGGCAAAAAAGCACCTGGAGATCAACCCTGACCACCCTATCGTGGAGACCCTGCGCCAGAAGGCTGAGGCTGACAAGAACGACAAGGCTGTGAAGGACCTGGTGGTGCTGCTGTTCGAGACCGCTCTGCTGTCCTCCGG TTTCTCCCTGGAGGACCCTCAGACCCACTCCAACCGCATCTACCGCATGATCAAGCTGGGTCTGGGTATCGACGAGGACGAGGTGACCGCTGAGGAGCCTTCCGCTGCTGTGCCTGACGAGATCCCTCCTCTGGAGGGTGACGAGGACGCTTCCCGCATGGAGGAGGTGGAC

SEQ ID No. 9 (mXAP2-DNA)

ATGGCCGATCTGATTGCTCGCTTGCGTGAGGACGGTATTCAGAAGAGGGTGATCCAGGAGGGTCGCGGTGAATTGCCTGACTTCCAGGATGGTACAAAAGCCACCTTCCACTTCCGCACCTTGCACTCTGACAACGAGGGTAGTGTGATCGATGACAGCAGAACGAGGGGCAAACCTATGGAGTTGATCGTGGGTAAAAAGTTCAAACTGCCCGTGTGGGAGACCATCGTGTGTACTATGCGCGAGGGTGAGATCGCTCAGTTCTTGTGCGACATCAAGCATGTGGTGCTGTACCCTCTGGTGGCGAAGTCTCTCCGTAACATCGCTGAAGGTAAGGACCCACTGGAGGGTCAACGCCATTGTTGTGGAATCGCTCAAATGCACGAGCACAGCTCCCTCGGTCACGCTGATTTGGACGCTCTGCAGCAGAACCCACAACCGTTGATCTTCCACATCGAAATGCTGAAGGTGGAATCCCCTGGAACTTACCAGCAGGACCCCTGGGCTATGACCGATGAAGAGAAGGCTAAAGCAGTGCCTGTGATCCACCAAGAAGGTAACCGCCTGTACCGCGAGGGTCAAGTTAAGGAGGCTGCCGCTAAATACTACGACGCTATCGCTTGTTTGAAGAACCTGCAGATGAAGGAGCAGCCTGGTAGCCCTGACTGGATTCAACTGGATCTGCAGATTACACCTCTCCTGCTGAACTACTGTCAGTGCAAGCTGGTGGCTCAGGAATACTACGAGGTGCTGGATCACTGCAGCTCCATCCTGAACAAATACGACGACAACGTGAAAGCTTACTTCAAACGCGGTAAAGCTCACGCTGCTGTCTGGAACGCTCAGGAAGCTCAGGCTGACTTCGCTAAAGTGCTCGAACTCGACCCCGCCTTGGCACCTGTTGTTTCAAGGGAGTTGCGCGCTCTCGAAACTCGCATCCGTCAGAAAGATGAGGAGGACAAGGCTCGTTTCCGCGGAATCTTCTCCCAC

SEQ ID No. 10 (mP23-DNA)

ATGCAGCCTGCCTCCGCTAAGTGGTACGACAGAAGGGACTACGTGTTCATCGAGTTCTGCGTGGAGGACTCCAAGGACGTGAACGTGAACTTCGAGAAGTCCAAATTGACCTTCAGCTGCCTGGGCGGTTCCGATAACTTCAAGCACCTGAACGAAATCGACCTCTTCCATTGTATCGATCCAAACGACTCCAAACACAAGAGGACTGACCGCTCCATCCTGTGCTGCTTGCGTAAGGGTGAGTCTGGTCAGTCCTGGCCAAGACTGACAAAGGAGAGAGCTAAGCTCAACTGGCTGTCCGTGGACTTCAACAACTGGAAAGACTGGGAAGACGACTCCGACGAGGACATGAGCAACTTCGACAGGTTCAGTGAGATGATGGACCACATGGGAGGAGATGAAGACGTGGATCTGCCTGAGGTGGACGGAGCTGATGACGATAGCCAGGACAGCGACGACGAGAAGATGCCAGACCTGGAG

SEQ ID No. 11

CTATGCATCAGCTGCTAGCACCATGGCTCGAATGGGCGCTTGGAGCCACCCTCAGTTCG

SEQ ID No. 12

TCGACGAAGACTTGATCACCCGGGATCGAAAGGCAGGGAGGTGCTCCTCTTCTG

SEQ ID No. 13

GCCTACGTCGACGAGCTCACTAGTCGCGGCCATGCCTGAGGAGGTGCACCACGGTGAAG

SEQ ID No. 14

GAGACTGCAGGCTCTAGATTCGAAAGCGTCCACCTCCTCCATGCGGGAAGCGTC

SEQ ID No. 15

CTATGCATCAGCTGCTAGCACCATGGCTCGAATGGCCGATCTGATTGCTCGCTTGCGTG

SEQ ID No. 16

TCGACGAAGACTTGATCACCCGGGATCGTGGGAGAAGATTCCGCGGAAACGAGC

SEQ ID No. 17

GCCTACGTCGACGAGCTCACTAGTCGCGGCCATGCAGCCTGCCTCCGCTAAGTGGTACG

SEQ ID No. 18

GAGACTGCAGGCTCTAGATTCGAAAGCCTCCAGGTCTGGCATCTTCTCGTCGTC

sequence listing

<110> Institute of Biophysics, Chinese Academy of Sciences

<120> A method for stable and high expression of aryl hydrocarbon receptor protein and its application

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 453

<212> PRT

<213>hAhR-1

<400> 1

Val Lys Pro Ile Pro Ala Glu Gly Ile Lys Ser Asn Pro Ser Lys Arg

1 5 10 15

His Arg Asp Arg Leu Asn Thr Glu Leu Asp Arg Leu Ala Ser Leu Leu

20 25 30

Pro Phe Pro Gln Asp Val Ile Asn Lys Leu Asp Lys Leu Ser Val Leu

35 40 45

Arg Leu Ser Val Ser Tyr Leu Arg Ala Lys Ser Phe Phe Asp Val Ala

50 55 60

Leu Lys Ser Ser Pro Thr Glu Arg Asn Gly Gly Gln Asp Asn Cys Arg

65 70 75 80

Ala Ala Asn Phe Arg Glu Gly Leu Asn Leu Gln Glu Gly Glu Phe Leu

85 90 95

Leu Gln Ala Leu Asn Gly Phe Val Leu Val Val Thr Thr Asp Ala Leu

100 105 110

Val Phe Tyr Ala Ser Ser Thr Ile Gln Asp Tyr Leu Gly Phe Gln Gln

115 120 125

Ser Asp Val Ile His Gln Ser Val Tyr Glu Leu Ile His Thr Glu Asp

130 135 140

Arg Ala Glu Phe Gln Arg Gln Leu His Trp Ala Leu Asn Pro Ser Gln

145 150 155 160

Cys Thr Glu Ser Gly Gln Gly Ile Glu Glu Ala Thr Gly Leu Pro Gln

165 170 175

Thr Val Val Cys Tyr Asn Pro Asp Gln Ile Pro Pro Glu Asn Ser Pro

180 185 190

Leu Met Glu Arg Cys Phe Ile Cys Arg Leu Arg Cys Leu Leu Asp Asn

195 200 205

Ser Ser Gly Phe Leu Ala Met Asn Phe Gln Gly Lys Leu Lys Tyr Leu

210 215 220

His Gly Gln Lys Lys Lys Lys Gly Lys Asp Gly Ser Ile Leu Pro Pro Gln

225 230 235 240

Leu Ala Leu Phe Ala Ile Ala Thr Pro Leu Gln Pro Pro Ser Ile Leu

245 250 255

Glu Ile Arg Thr Lys Asn Phe Ile Phe Arg Thr Lys His Lys Leu Asp

260 265 270

Phe Thr Pro Ile Gly Cys Asp Ala Lys Gly Arg Ile Val Leu Gly Tyr

275 280 285

Thr Glu Ala Glu Leu Cys Thr Arg Gly Ser Gly Tyr Gln Phe Ile His

290 295 300

Ala Ala Asp Met Leu Tyr Cys Ala Glu Ser His Ile Arg Met Ile Lys

305 310 315 320

Thr Gly Glu Ser Gly Met Ile Val Phe Arg Leu Leu Thr Lys Asn Asn

325 330 335

Arg Trp Thr Trp Val Gln Ser Asn Ala Arg Leu Leu Tyr Lys Asn Gly

340 345 350

Arg Pro Asp Tyr Ile Ile Val Thr Gln Arg Pro Leu Thr Asp Glu Glu

355 360 365

Gly Thr Glu His Leu Arg Lys Arg Asn Thr Lys Leu Pro Phe Met Phe

370 375 380

Thr Thr Gly Glu Ala Val Leu Tyr Glu Ala Thr Asn Pro Phe Pro Ala

385 390 395 400

Ile Met Asp Pro Leu Pro Leu Arg Thr Lys Asn Gly Thr Ser Gly Lys

405 410 415

Asp Ser Ala Thr Thr Ser Thr Leu Ser Lys Asp Ser Leu Asn Pro Ser

420 425 430

Ser Leu Leu Ala Ala Met Met Met Gln Gln Asp Glu Ser Ile Tyr Leu Tyr

435 440 445

Pro Ala Ser Ser Thr

450

<210> 2

<211> 398

<212> PRT

<213> mAhR-1

<400> 2

Met Ser Ser Gly Ala Asn Ile Thr Tyr Ala Ser Arg Lys Arg Arg Lys

1 5 10 15

Pro Val Gln Lys Thr Val Lys Pro Ile Pro Ala Glu Gly Ile Lys Ser

20 25 30

Asn Pro Ser Lys Arg His Arg Asp Arg Leu Asn Thr Glu Leu Asp Arg

35 40 45

Leu Ala Ser Leu Leu Pro Phe Pro Gln Asp Val Ile Asn Lys Leu Asp

50 55 60

Lys Leu Ser Val Leu Arg Leu Ser Val Ser Tyr Leu Arg Ala Lys Ser

65 70 75 80

Phe Phe Asp Val Ala Leu Lys Ser Thr Pro Ala Asp Arg Asn Gly Gly

85 90 95

Gln Asp Gln Cys Arg Ala Gln Ile Arg Asp Trp Gln Asp Leu Gln Glu

100 105 110

Gly Glu Phe Leu Leu Gln Ala Leu Asn Gly Phe Val Leu Val Val Thr

115 120 125

Ala Asp Ala Leu Val Phe Tyr Ala Ser Ser Thr Ile Gln Asp Tyr Leu

130 135 140

Gly Phe Gln Gln Ser Asp Val Ile His Gln Ser Val Tyr Glu Leu Ile

145 150 155 160

His Thr Glu Asp Arg Ala Glu Phe Gln Arg Gln Leu His Trp Ala Leu

165 170 175

Asn Pro Asp Ser Ala Gln Gly Val Asp Glu Ala His Gly Pro Pro Gln

180 185 190

Ala Ala Val Tyr Tyr Thr Pro Asp Gln Leu Pro Pro Glu Asn Ala Ser

195 200 205

Phe Met Glu Arg Cys Phe Arg Cys Arg Leu Arg Cys Leu Leu Asp Asn

210 215 220

Ser Ser Gly Phe Leu Ala Met Asn Phe Gln Gly Arg Leu Lys Tyr Leu

225 230 235 240

His Gly Gln Asn Lys Lys Lys Gly Lys Asp Gly Ala Leu Leu Pro Pro Gln

245 250 255

Leu Ala Leu Phe Ala Ile Ala Thr Pro Leu Gln Pro Pro Ser Ile Leu

260 265 270

Glu Ile Arg Thr Lys Asn Phe Ile Phe Arg Thr Lys His Lys Leu Asp

275 280 285

Phe Thr Pro Ile Gly Cys Asp Ala Lys Gly Gln Leu Ile Leu Gly Tyr

290 295 300

Thr Glu Val Glu Leu Cys Thr Arg Gly Ser Gly Tyr Gln Phe Ile His

305 310 315 320

Ala Ala Asp Met Leu His Cys Ala Glu Ser His Ile Arg Met Ile Lys

325 330 335

Thr Gly Glu Ser Gly Met Thr Val Phe Arg Leu Phe Ala Lys His Ser

340 345 350

Arg Trp Arg Trp Val Gln Ser Asn Ala Arg Leu Ile Tyr Arg Asn Gly

355 360 365

Arg Pro Asp Tyr Ile Ile Ala Thr Gln Arg Pro Leu Thr Asp Glu Glu

370 375 380

Gly Arg Glu His Leu Gln Lys Arg Ser Thr Ser Leu Pro Phe

385 390 395

<210> 3

<211> 724

<212> PRT

<213> mHsp90

<400> 3

Met Pro Glu Glu Val His His Gly Glu Glu Glu Val Glu Thr Phe Ala

1 5 10 15

Phe Gln Ala Glu Ile Ala Gln Leu Met Ser Leu Ile Ile Asn Thr Phe

20 25 30

Tyr Ser Asn Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser

35 40 45

Asp Ala Leu Asp Lys Ile Arg Tyr Glu Ser Leu Thr Asp Pro Ser Lys

50 55 60

Leu Asp Ser Gly Lys Glu Leu Lys Ile Asp Ile Ile Pro Asn Pro Gln

65 70 75 80

Glu Arg Thr Leu Thr Leu Val Asp Thr Gly Ile Gly Met Thr Lys Ala

85 90 95

Asp Leu Ile Asn Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Lys Ala

100 105 110

Phe Met Glu Ala Leu Gln Ala Gly Ala Asp Ile Ser Met Ile Gly Gln

115 120 125

Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val Ala Glu Lys Val Val

130 135 140

Val Ile Thr Lys His Asn Asp Asp Glu Gln Tyr Ala Trp Glu Ser Ser

145 150 155 160

Ala Gly Gly Ser Phe Thr Val Arg Ala Asp His Gly Glu Pro Ile Gly

165 170 175

Arg Gly Thr Lys Val Ile Leu His Leu Lys Glu Asp Gln Thr Glu Tyr

180 185 190

Leu Glu Glu Arg Arg Val Lys Glu Val Val Lys Lys His Ser Gln Phe

195 200 205

Ile Gly Tyr Pro Ile Thr Leu Tyr Leu Glu Lys Glu Arg Glu Lys Glu

210 215 220

Ile Ser Asp Asp Glu Ala Glu Glu Glu Lys Gly Glu Lys Glu Glu Glu

225 230 235 240

Asp Lys Glu Asp Glu Glu Lys Pro Lys Ile Glu Asp Val Gly Ser Asp

245 250 255

Glu Glu Asp Asp Ser Gly Lys Asp Lys Lys Lys Lys Lys Thr Lys Lys Ile

260 265 270

Lys Glu Lys Tyr Ile Asp Gln Glu Glu Leu Asn Lys Thr Lys Pro Ile

275 280 285

Trp Thr Arg Asn Pro Asp Asp Ile Thr Gln Glu Glu Tyr Gly Glu Phe

290 295 300

Tyr Lys Ser Leu Thr Asn Asp Trp Glu Asp His Leu Ala Val Lys His

305 310 315 320

Phe Ser Val Glu Gly Gln Leu Glu Phe Arg Ala Leu Leu Phe Ile Pro

325 330 335

Arg Arg Ala Pro Phe Asp Leu Phe Glu Asn Lys Lys Lys Lys Asn Asn

340 345 350

Ile Lys Leu Tyr Val Arg Arg Val Phe Ile Met Asp Ser Cys Asp Glu

355 360 365

Leu Ile Pro Glu Tyr Leu Asn Phe Ile Arg Gly Val Val Asp Ser Glu

370 375 380

Asp Leu Pro Leu Asn Ile Ser Arg Glu Met Leu Gln Gln Ser Lys Ile

385 390 395 400

Leu Lys Val Ile Arg Lys Asn Ile Val Lys Lys Cys Leu Glu Leu Phe

405 410 415

Ser Glu Leu Ala Glu Asp Lys Glu Asn Tyr Lys Lys Phe Tyr Glu Ala

420 425 430

Phe Ser Lys Asn Leu Lys Leu Gly Ile His Glu Asp Ser Thr Asn Arg

435 440 445

Arg Arg Leu Ser Glu Leu Leu Arg Tyr His Thr Ser Gln Ser Gly Asp

450 455 460

Glu Met Thr Ser Leu Ser Glu Tyr Val Ser Arg Met Lys Glu Thr Gln

465 470 475 480

Lys Ser Ile Tyr Tyr Ile Thr Gly Glu Ser Lys Glu Gln Val Ala Asn

485 490 495

Ser Ala Phe Val Glu Arg Val Arg Lys Arg Gly Phe Glu Val Val Tyr

500 505 510

Met Thr Glu Pro Ile Asp Glu Tyr Cys Val Gln Gln Leu Lys Glu Phe

515 520 525

Asp Gly Lys Ser Leu Val Ser Val Thr Lys Glu Gly Leu Glu Leu Pro

530 535 540

Glu Asp Glu Glu Glu Lys Lys Lys Met Glu Glu Ser Lys Ala Lys Phe

545 550 555 560

Glu Asn Leu Cys Lys Leu Met Lys Glu Ile Leu Asp Lys Lys Val Glu

565 570 575

Lys Val Thr Ile Ser Asn Arg Leu Val Ser Ser Pro Cys Cys Ile Val

580 585 590

Thr Ser Thr Tyr Gly Trp Thr Ala Asn Met Glu Arg Ile Met Lys Ala

595 600 605

Gln Ala Leu Arg Asp Asn Ser Thr Met Gly Tyr Met Met Ala Lys Lys

610 615 620

His Leu Glu Ile Asn Pro Asp His Pro Ile Val Glu Thr Leu Arg Gln

625 630 635 640

Lys Ala Glu Ala Asp Lys Asn Asp Lys Ala Val Lys Asp Leu Val Val

645 650 655

Leu Leu Phe Glu Thr Ala Leu Leu Ser Ser Gly Phe Ser Leu Glu Asp

660 665 670

Pro Gln Thr His Ser Asn Arg Ile Tyr Arg Met Ile Lys Leu Gly Leu

675 680 685

Gly Ile Asp Glu Asp Glu Val Thr Ala Glu Glu Pro Ser Ala Ala Val

690 695 700

Pro Asp Glu Ile Pro Pro Leu Glu Gly Asp Glu Asp Ala Ser Arg Met

705 710 715 720

Glu Glu Val Asp

<210> 4

<211> 330

<212> PRT

<213> mXAP2

<400> 4

Met Ala Asp Leu Ile Ala Arg Leu Arg Glu Asp Gly Ile Gln Lys Arg

1 5 10 15

Val Ile Gln Glu Gly Arg Gly Glu Leu Pro Asp Phe Gln Asp Gly Thr

20 25 30

Lys Ala Thr Phe His Phe Arg Thr Leu His Ser Asp Asn Glu Gly Ser

35 40 45

Val Ile Asp Asp Ser Arg Thr Arg Gly Lys Pro Met Glu Leu Ile Val

50 55 60

Gly Lys Lys Phe Lys Leu Pro Val Trp Glu Thr Ile Val Cys Thr Met

65 70 75 80

Arg Glu Gly Glu Ile Ala Gln Phe Leu Cys Asp Ile Lys His Val Val

85 90 95

Leu Tyr Pro Leu Val Ala Lys Ser Leu Arg Asn Ile Ala Glu Gly Lys

100 105 110

Asp Pro Leu Glu Gly Gln Arg His Cys Cys Gly Ile Ala Gln Met His

115 120 125

Glu His Ser Ser Leu Gly His Ala Asp Leu Asp Ala Leu Gln Gln Asn

130 135 140

Pro Gln Pro Leu Ile Phe His Ile Glu Met Leu Lys Val Glu Ser Pro

145 150 155 160

Gly Thr Tyr Gln Gln Asp Pro Trp Ala Met Thr Asp Glu Glu Lys Ala

165 170 175

Lys Ala Val Pro Val Ile His Gln Glu Gly Asn Arg Leu Tyr Arg Glu

180 185 190

Gly Gln Val Lys Glu Ala Ala Ala Lys Tyr Tyr Asp Ala Ile Ala Cys

195 200 205

Leu Lys Asn Leu Gln Met Lys Glu Gln Pro Gly Ser Pro Asp Trp Ile

210 215 220

Gln Leu Asp Leu Gln Ile Thr Pro Leu Leu Leu Asn Tyr Cys Gln Cys

225 230 235 240

Lys Leu Val Ala Gln Glu Tyr Tyr Glu Val Leu Asp His Cys Ser Ser

245 250 255

Ile Leu Asn Lys Tyr Asp Asp Asn Val Lys Ala Tyr Phe Lys Arg Gly

260 265 270

Lys Ala His Ala Ala Val Trp Asn Ala Gln Glu Ala Gln Ala Asp Phe

275 280 285

Ala Lys Val Leu Glu Leu Asp Pro Ala Leu Ala Pro Val Val Ser Arg

290 295 300

Glu Leu Arg Ala Leu Glu Thr Arg Ile Arg Gln Lys Asp Glu Glu Asp

305 310 315 320

Lys Ala Arg Phe Arg Gly Ile Phe Ser His

325 330

<210> 5

<211> 160

<212> PRT

<213> mP23

<400> 5

Met Gln Pro Ala Ser Ala Lys Trp Tyr Asp Arg Arg Asp Tyr Val Phe

1 5 10 15

Ile Glu Phe Cys Val Glu Asp Ser Lys Asp Val Asn Val Asn Phe Glu

20 25 30

Lys Ser Lys Leu Thr Phe Ser Cys Leu Gly Gly Ser Asp Asn Phe Lys

35 40 45

His Leu Asn Glu Ile Asp Leu Phe His Cys Ile Asp Pro Asn Asp Ser

50 55 60

Lys His Lys Arg Thr Asp Arg Ser Ile Leu Cys Cys Leu Arg Lys Gly

65 70 75 80

Glu Ser Gly Gln Ser Trp Pro Arg Leu Thr Lys Glu Arg Ala Lys Leu

85 90 95

Asn Trp Leu Ser Val Asp Phe Asn Asn Trp Lys Asp Trp Glu Asp Asp

100 105 110

Ser Asp Glu Asp Met Ser Asn Phe Asp Arg Phe Ser Glu Met Met Asp

115 120 125

His Met Gly Gly Asp Glu Asp Val Asp Leu Pro Glu Val Asp Gly Ala

130 135 140

Asp Asp Asp Ser Gln Asp Ser Asp Asp Glu Lys Met Pro Asp Leu Glu

145 150 155 160

<210> 6

<211> 1359

<212> DNA

<213> hAhR-1-DNA

<400> 6

gtaaaaccta ttccggcaga aggcattaaa agcaacccga gcaaaagaca tcgtgaccgg 60

ttaaatacag agctggatcg tctggcaagc cttttaccgt tcccgcagga tgttattaat 120

aaactggaca aactgagtgt actgcgcctg agcgttagct atttacgtgc taaaagtttt 180

ttcgacgtgg ctctgaaatc tagtccgacc gagcgtaatg gtgggcagga taactgccgg 240

gcagcaaact ttcgcgaagg tctgaattta caggaggggg aatttcttct gcaggctctg 300

aatggttttg ttctggttgt tacaaccgat gcactggttt tttatgcaag ctctacaatt 360

caggattatt tagggtttca acagagcgat gtgatacatc agtctgttta tgaattaatc 420

cacaccgagg accgtgcaga gtttcaaaga caactgcatt gggcactgaa cccaagccag 480

tgtaccgaga gcgggcaagg tatcgaggaa gcaaccggtc ttccacagac cgtggtttgt 540

tataatccgg atcagattcc gccagaaaat agccccctga tggaacgctg ttttatttgt 600

cgcctgagat gtttactgga caatagctca ggctttctgg caatgaactt tcagggtaaa 660

ctgaaatatc ttcacggtca gaagaaaaag ggtaaagatg gaagtattct gccgccgcaa 720

cttgcactgt ttgcaattgc aacccccctg cagcctccgt ctatcctgga aattcgcacc 780

aaaaatttta tctttcgtac caaacacaag ctggatttta cccccattgg ttgtgatgcc 840

aaaggtcgta ttgttttagg ctataccgaa gcagaactgt gcacccgggg ttcaggctat 900

cagtttattc atgcagcaga tatgctgtat tgtgccgaaa gccacattcg gatgattaag 960

acaggtgaaa gtgggatgat tgtttttcgt ctgcttacca aaaataaccg ttggacctgg 1020

gtgcagagca atgcacgttt attatataag aatggtcgtc cggactatat tattgttaca 1080

cagcggccgt taaccgacga ggagggtacc gaacatctgc gtaaacgcaa caccaaatta 1140

ccatttatgt ttaccaccgg tgaggcggtg ctgtatgaag caaccaaccc gtttccggca 1200

attatggacc ctttaccgct gcgtaccaaa aacggtacct caggtaaaga cagcgcgacc 1260

accagcacct taagcaaaga tagcctgaat cccagcagtc tgctggcagc tatgatgcag 1320

caggatgaaa gcatttatct gtatccagca agcagcacc 1359

<210> 7

<211> 1194

<212> DNA

<213>mAhR-1-DNA

<400> 7

atgagcagtg gagctaacat tacttacgcc tccaggaaga gaaggaagcc tgttcagaag 60

actgtgaagc caatccctgc cgagggtatt aaatccaacc cctccaagcg ccacagggac 120

agattgaaca ccgagctgga ccgcctggct agtttgttgc cattccccca ggacgtgatc 180

aacaagctcg acaaactgtc cgtgctgcgc ctgtctgtct cctacttgcg tgcaaagtcc 240

ttcttcgacg tggctttgaa atccaccccc gctgaccgta acggtggtca agatcaatgc 300

cgcgctcaga tccgcgactg gcaagatttg caggagggtg agttcctcct gcaggctttg 360

aacggattcg tgttggtggt caccgctgac gctctggttt tctacgcttc ctccaccatt 420

caggactacc tgggattcca acagagcgac gtgattcatc agtccgttta cgagctgatc 480

cacaccgagg accgcgctga atttcagcgt caattgcact gggctctgaa ccctgacagt 540

gctcagggtg tggacgaggc tcatggtcct cctcaagcag ctgtgtacta cacccccgac 600

cagttgcctc cagaaaacgc ttccttcatg gagcgctgtt tccgctgccg tctgagatgt 660

ctcctggata actcctccgg tttcctcgct atgaacttcc agggtcgcct gaagtacctc 720

cacggtcaaa acaaaaaggg taaggacgga gctttgctcc ctccccaact ggctttgttc 780

gctattgcca cccctctgca gcctccttct atcttggaaa ttcgcaccaa gaacttcatc 840

ttccgcacca agcacaagct cgacttcacc cctatcggtt gcgacgctaa gggtcagctg 900

atcctgggat acaccgaagt cgagctgtgc acccgtggtt ctggatacca attcatccac 960

gctgccgaca tgctgcactg cgctgaatct cacatccgta tgatcaagac tggtgagtcc 1020

ggtatgaccg tgttccgctt gttcgctaag cattctcgct ggcgctgggt ccaatcaaac 1080

gctagactga tctaccgtaa cggtcgcccc gactacatca tcgcaaccca aagacctctg 1140

accgacgagg agggtaggga acatctgcag aagaggagca cctccctgcc tttc 1194

<210> 8

<211> 2172

<212> DNA

<213> mHsp90-DNA

<400> 8

atgcctgagg aggtgcacca cggtgaagaa gaagtggaaa ccttcgcttt ccaggctgag 60

attgctcaac tgatgtccct catcatcaac accttctaca gcaacaagga gatcttcctg 120

agagagctga tcagcaacgc ttccgacgct ctggacaaaa tccgttacga gtccctgacc 180

gatccttcca aactggactc cggcaaagaa ctgaagatcg acatcatccc taaccctcag 240

gagcgcaccc tcacattggt ggataccggt atcggcatga caaaggcaga cctcatcaac 300

aacctgggca ccattgctaa aagcggaacc aaagccttca tggaggctct gcaagcaggt 360

gctgacatct ccatgattgg tcaattcggc gtgggtttct actccgctta cctcgtggcc 420

gagaaggtgg ttgtgatcac aaagcacaac gacgacgagc agtacgcttg ggagagcagt 480

gctggtggtt ccttcactgt cagggctgat cacggtgagc ctatcggtcg tggtaccaaa 540

gtgatcctgc atctgaagga ggaccaaacc gagtacctcg aagagcgcag ggtgaaggag 600

gtggttaaga agcacagcca gttcatcggt taccccatca cactgtactt ggagaaagag 660

cgcgaaaaag agatcagcga cgacgaagca gaggaagaaa agggtgagaa ggaagaggaa 720

gataaggagg acgaggagaa gccaaagatc gaggacgtgg gaagcgacga ggaggatgat 780

agcggaaagg acaagaagaa gaagacaaag aagatcaagg agaagtacat cgaccaggag 840

gagctgaaca agacaaagcc aatctggaca aggaaccctg acgacatcac acaggaggag 900

tacggagagt tctacaagag cctcacaaac gactgggagg accacctggc tgtgaagcat 960

ttcagcgtgg agggacagct ggagttcagg gctttgctgt tcatcccaag gagagcccct 1020

ttcgacctgt tcgagaacaa aaagaagaag aacaacatca aactgtacgt gcgccgcgtc 1080

ttcattatgg actcctgcga cgagctgatc cctgagtacc tgaacttcat ccgtggtgtg 1140

gtggactccg aagacctgcc tttgaacatc tcccgcgaga tgctgcagca gtccaagatc 1200

ctgaaggtga tcaggaagaa catcgtgaag aaatgcctgg agttgttctc cgagttggct 1260

gaagacaaag agaactacaa gaagttctac gaggcattct ccaagaacct gaagctgggt 1320

atccatgagg actccaccaa ccgccgtcgt ttgtctgagc tgctgcgtta ccacaccagc 1380

cagtctggtg acgagatgac ctccctgtcc gaatacgtgt cccgcatgaa ggaaactcag 1440

aaatcaattt actacatcac tggtgaatcc aaggagcaag tggctaactc cgctttcgtc 1500

gagcgtgtgc gtaagagagg tttcgaggtg gtgtacatga ccgagcccat cgacgaatac 1560

tgcgtccagc agctgaagga gttcgacggt aagtccttgg tgagcgtcac caaggagggt 1620

ctggagttgc cagaggatga ggaggagaag aagaagatgg aagagagcaa ggcaaagttc 1680

gagaacctct gcaagttgat gaaggagatc ctggacaaga aggtggagaa ggtgactatc 1740

agcaacaggc tggtgagcag tccatgctgc atcgtgacca gcacctacgg atggaccgca 1800

aacatggaga ggatcatgaa ggcacaagcc ctgcgcgaca acagcacaat gggatacatg 1860

atggcaaaaa agcacctgga gatcaaccct gaccacccta tcgtggagac cctgcgccag 1920

aaggctgagg ctgacaagaa cgacaaggct gtgaaggacc tggtggtgct gctgttcgag 1980

accgctctgc tgtcctccgg tttctccctg gaggaccctc agacccactc caaccgcatc 2040

taccgcatga tcaagctggg tctgggtatc gacgaggacg aggtgaccgc tgaggagcct 2100

tccgctgctg tgcctgacga gatccctcct ctggagggtg acgaggacgc ttcccgcatg 2160

gaggaggtgg ac 2172

<210> 9

<211> 990

<212> DNA

<213> mXAP2-DNA

<400> 9

atggccgatc tgattgctcg cttgcgtgag gacggtattc agaagagggt gatccaggag 60

ggtcgcggtg aattgcctga cttccaggat ggtacaaaag ccaccttcca cttccgcacc 120

ttgcactctg acaacgaggg tagtgtgatc gatgacagca gaacgagggg caaacctatg 180

gagttgatcg tgggtaaaaa gttcaaactg cccgtgtggg agaccatcgt gtgtactatg 240

cgcgagggtg agatcgctca gttcttgtgc gacatcaagc atgtggtgct gtaccctctg 300

gtggcgaagt ctctccgtaa catcgctgaa ggtaaggacc cactggaggg tcaacgccat 360

tgttgtggaa tcgctcaaat gcacgagcac agctccctcg gtcacgctga tttggacgct 420

ctgcagcaga acccacaacc gttgatcttc cacatcgaaa tgctgaaggt ggaatcccct 480

ggaacttacc agcaggaccc ctgggctatg accgatgaag agaaggctaa agcagtgcct 540

gtgatccacc aagaaggtaa ccgcctgtac cgcgagggtc aagttaagga ggctgccgct 600

aaatactacg acgctatcgc ttgtttgaag aacctgcaga tgaaggagca gcctggtagc 660

cctgactgga ttcaactgga tctgcagatt acacctctcc tgctgaacta ctgtcagtgc 720

aagctggtgg ctcaggaata ctacgaggtg ctggatcact gcagctccat cctgaacaaa 780

tacgacgaca acgtgaaagc ttacttcaaa cgcggtaaag ctcacgctgc tgtctggaac 840

gctcaggaag ctcaggctga cttcgctaaa gtgctcgaac tcgaccccgc cttggcacct 900

gttgtttcaa gggagttgcg cgctctcgaa actcgcatcc gtcagaaaga tgaggaggac 960

aaggctcgtt tccgcggaat cttctcccac 990

<210> 10

<211> 480

<212> DNA

<213> mP23-DNA

<400> 10

atgcagcctg cctccgctaa gtggtacgac agaagggact acgtgttcat cgagttctgc 60

gtggaggact ccaaggacgt gaacgtgaac ttcgagaagt ccaaattgac cttcagctgc 120

ctgggcggtt ccgataactt caagcacctg aacgaaatcg acctcttcca ttgtatcgat 180

ccaaacgact ccaaacacaa gaggactgac cgctccatcc tgtgctgctt gcgtaagggt 240

gagtctggtc agtcctggcc aagactgaca aaggagag ctaagctcaa ctggctgtcc 300

gtggacttca acaactggaa agactgggaa gacgactccg acgaggacat gagcaacttc 360

gacaggttca gtgagatgat ggaccacatg ggaggagatg aagacgtgga tctgcctgag 420

gtggacggag ctgatgacga tagccaggac agcgacgacg agaagatgcc agacctggag 480

Claims (10)

1. A method for stably and efficiently expressing an aromatic hydrocarbon receptor protein is characterized by comprising the following steps: the stable and efficient expression of AhR can be realized by utilizing molecular chaperone to assist the co-expression of AhR protein in the expression of aromatic hydrocarbon receptor protein (AhR); wherein the mass ratio of the aromatic hydrocarbon receptor protein (AhR) to the molecular chaperone is 1: 1.

2. The method for stable and efficient expression of an aromatic hydrocarbon receptor protein according to claim 1, wherein: the AhR protein is an amino acid sequence comprising a PAS-B domain.

3. The method for stable and efficient expression of an aromatic hydrocarbon receptor protein according to claim 2, wherein: the AhR protein is human AhR or murine AhR; the molecular chaperone is one or more of Hsp90, XAP2 and p23 of human or mouse sources.

4. The method for stable and efficient expression of an aromatic hydrocarbon receptor protein according to any one of claims 1 to 5, wherein: the molecular chaperone and the AhR protein are jointly constructed on the same vector for joint expression; or, the molecular chaperone and the AhR protein are respectively constructed on the same carrier, and then the co-transfection host cell is co-expressed; or adding purified molecular chaperone protein and AhR protein into the expression system for co-expression.

5. An aromatic hydrocarbon receptor protein with stable and high-efficiency expression, which is characterized in that: the method of claim 1, wherein the aromatic hydrocarbon receptor protein is stably and efficiently expressed.

6. The use of the aromatic hydrocarbon receptor protein stably and efficiently expressed according to the method of claim 1, wherein: the method of claim 1, wherein the aromatic hydrocarbon receptor protein is stably and efficiently expressed for detection of pollutants.

7. An expression vector for detecting pollutant dioxin-like chemical substances is characterized in that: the vector contains the aromatic hydrocarbon receptor protein which is stably and efficiently expressed and obtained by the method of claim 1.

8. The expression vector of claim 7, wherein: the molecular chaperone and the AhR protein are respectively or jointly constructed on the same vector for co-expression, wherein the vector is a pFastBac-Dual vector, a pETDuet-1 vector and a pBudCE4.1 vector.

9. A host cell for detecting a contaminant dioxin-like chemical, comprising: the host cell contains the aromatic hydrocarbon receptor protein which is stably and efficiently expressed and obtained by the method of claim 1.

10. The host cell of claim 9, wherein: the host cell is insect cell, colibacillus or yeast.

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