Method for separating and purifying peroxisome

Abstract

The invention relates to the technical field of organelle separation, and provides a combination of ATF6 alpha N protein and peroxisome, application of the ATF6 alpha N protein in separation and purification of the peroxisome and a method for separating and purifying the peroxisome. The ATF6 alpha N protein is combined with membrane protein PMP70 and / or ABCD1 of the peroxisome; the method comprises the following steps: combining tagne-ATF6 alpha N fusion protein with a medium to obtain a fusion protein-medium compound; adding a roughly separated peroxisome sample into the fusion protein-medium compound, so as to obtain a fusion protein-medium-peroxisome compound; and adding an elution buffer solution, and collecting the eluent to obtain the peroxisome combined with the fusion protein. The method is easy and convenient to operate, complex instruments and equipment are not needed, the peroxisome can be separated from cells and tissues, and the obtained peroxisome is high in purity and can be used for conventional experiments such as subsequent protein spectrum analysis and flow analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of organelle separation, and particularly relates to a method for separating and purifying peroxisomes. Background Art

[0002] There are various organelles in cells. There are both functional synergies and physical interactions among various organelles. Peroxisome is a tiny organelle in the cytoplasm, with a diameter of about 0.1 - 1 μm, and it is an important site where key metabolic activities such as α and β oxidation of fatty acids and oxidative stress occur in cells. It has been reported that peroxisomes interact with organelles such as mitochondria, endoplasmic reticulum, and lysosomes.

[0003] Peroxisomes are closely related to the occurrence and development of various diseases such as tumorigenesis, sensitivity of tumors to chemotherapeutic drugs, antiviral infection, and Alzheimer's disease. A simple and easy peroxisome separation technology can not only provide technical means for studying the pathogenesis of various diseases, but also contribute to the development of tumor - targeted drugs targeting peroxisomes, and also help in the development of early diagnostic indicators for diseases such as tumors and Alzheimer's disease.

[0004] At the present stage, the technologies used for separating and purifying peroxisomes include density gradient centrifugation technology, immunoprecipitation method targeting peroxisomal membrane protein PMP70, free - flow electrophoresis, etc. For example, Manner A, Islinger M. Isolation of Mammalian Peroxisomes by Density Gradient Centrifugation. Methods Mol Biol, 2023, 2643: 1 - 12. discloses a protocol for purifying peroxisomes from liver tissue or HepG2 hepatoma cell line by density gradient centrifugation method. However, the existing technologies for separating and purifying peroxisomes may have problems such as cumbersome methods, high requirements for instrument equipment, insufficient purity, the isolated peroxisomes are usually suspended in the gradient solution, which is not conducive to carrying out subsequent experiments, and high costs. Summary of the Invention

[0005] In view of the above - mentioned disadvantages of the prior art, in the first aspect, the present invention provides a conjugate, which is a conjugate of ATF6αN protein and peroxisomes.

[0006] In the second aspect, the present invention provides the use of ATF6αN protein in separating and purifying peroxisomes.

[0007] In the third aspect, the present invention provides a method for separating and purifying peroxisomes, and the method comprises the following steps:

[0008] (1) The tag-ATF6αN fusion protein binds to the medium to obtain a fusion protein-medium complex, and the tag can bind to the medium;

[0009] (2) Add the crude peroxisome sample to the fusion protein-medium complex to obtain a fusion protein-medium-peroxisome complex;

[0010] (3) Add the elution buffer and collect the eluate to obtain peroxisomes bound with the tag-ATF6αN fusion protein.

[0011] The beneficial effects of the present invention are as follows:

[0012] The separation and purification method provided by the present invention can simply and efficiently isolate relatively pure peroxisomes. This method is easy to operate and does not require complex instruments and equipment. Peroxisomes can be isolated from any cells and tissues, and the obtained peroxisomes have a high purity and can be used for subsequent conventional experiments such as proteomic analysis and flow cytometry analysis. The separation and purification method provided by the present invention has broad application prospects in the research on peroxisome function and homeostasis, as well as the research on the interaction between organelles in cells. Brief Description of the Drawings

[0013] Figure 1 . The interaction between ATF6αN protein and peroxisomal membrane protein.

[0014] A. Transfect HeLa cells with a plasmid expressing GFP-ATF6αN protein for 24 hours, and then treat with DMSO or Ceapin-A7

[0015] (6 μM) for 24 hours, then fix the cells and stain with PMP70 (Alexa Fluor 555), scale bar: 5 μm.

[0016] B. Transfect HEK293T cells with a plasmid expressing GFP-ATF6αN protein for 24 hours, and then treat with DMSO or Ceapin-

[0017] A7 (6 μM) for 24 hours, immunoprecipitate the cell lysate with a GFP antibody, and perform immunoblotting with the indicated antibody.

[0018] C. Coomassie staining SDS-PAGE gel shows the expression of GST and GST-ATF6αN proteins.

[0019] D. Incubate HeLa cell lysate with purified GST or GST-ATF6αN protein, and then use DMSO or Ceapin-

[0020] The cells were treated with A7 (6 μM) for 2 hours, and the GST-tagged proteins were captured using glutathione affinity resin. Immunoblot analysis showed an interaction between GST-ATF6αN protein and PMP70, and this interaction was independent of the presence of Ceapin-A7.

[0021] E. GST-ATF6αN protein at increasing gradients was incubated with HeLa cell lysates, and then treated with DMSO or Ceapin-

[0022] A7 (6 μM) for 2 hours. Immunoblot analysis showed that the interaction between GST-ATF6αN protein and PMP70 was enhanced with the increasing amount of GST-ATF6αN protein added to the cell lysates.

[0023] F. HeLa cell lysates were incubated with purified GST or GST-ATF6αN protein, and the GST-tagged proteins were captured using glutathione affinity resin. Immunoblot analysis showed that GFP-ATF6αN protein interacted with both PMP70 and ABCD1.

[0024] G. Purified PMP70-Flag and GST-ATF6αN proteins were mixed, pulled down using Flag affinity gel, and then separated by IB for Flag and GST.

[0025] H. Purified Flag-ABCD1 and GST-ATF6αN proteins were mixed, pulled down using glutathione affinity resin, and then

[0026] pulled down by IB for Flag and GST.

[0027] I. Coomassie staining of SDS-PAGE gel showed that the interaction between Flag-ABCD1 and GST-ATF6αN proteins was at amino acids 2 - 90.

[0028] J-K. GST pull down experiments showed that GST-ATF6αN protein did not interact with either PEX19 or PEX5. Figure 2 . Design and validation of peroxisome isolation strategy.

[0029] A. Schematic diagram of the peroxisome isolation workflow of the present invention.

[0030] B. CSS cells, cell lysates, crude peroxisomes, and pure peroxisomes isolated from CSS cells by the method of Example 7 were analyzed using confocal microscopy. The scale bar for CSS is 2 μm. The scale bars for cell lysates, crude peroxisomes, and pure peroxisomes are 10 μm.

[0031] Quantification of fluorescence signals in C.B. The values are expressed as mean ± SD, n = 5.

[0032] D. Immunoblot analysis was used to evaluate the expression of organelle markers in peroxisomes isolated and purified from cell lysates of WT and PEX19 KO by the method of Example 7.

[0033] E - G. Catalase activity, glutamate dehydrogenase (GDH) activity, and carboxylesterase (CarE) activity were detected in peroxisomes isolated and purified from cell lysates of WT and PEX19 KO by the method of Example 7.

[0034] Figure 3 . Comparison with the traditional density gradient centrifugation method.

[0035] A. Representative fluorescence images of crude peroxisomes, peroxisomes isolated and purified in Example 7, and peroxisomes purified by density gradient centrifugation, with a scale bar of 20 μm.

[0036] B. Different organelles obtained by purification using traditional density gradient centrifugation and isolation and purification in Example 7 were analyzed by immunoblotting. C. Flow cytometry analysis of peroxisomes isolated and purified from WT and GFP - SKL cells in Example 7.

[0037] D. Peroxisomes isolated and purified from HeLa cells by the method of Example 7 were stained with PEX14 antibody (Alexa Fluor 555), and then analyzed by flow cytometry.

[0038] E. Peroxisomes isolated and purified from GFP - SKL cells by the method of Example 7 were stained with PEX14 antibody (Alexa Fluor 555), and then analyzed by flow cytometry.

[0039] F. Cryo - transmission electron microscopy showed the morphology of peroxisomes isolated and purified by the method of Example 7.

[0040] G. Proteomic identification of peroxisomes in HeLa cells.

[0041] Figure 4 A. Ratio - intensity plots of quantitatively proteomic proteins of peroxisomes isolated and purified by the method of Example 7 and whole - cell lysates.

[0042] B. Volcano plots of quantitatively proteomic proteins in peroxisomes isolated and purified from WT HeLa cells and PEX19 KO HeLa cells by the method of Example 7.

[0043] C.

[0044] C. Identification of 280 peroxisomal proteins identified from peroxisomal proteins isolated and purified from WT HeLa cells and PEX19 KO HeLa cells by the method of Example 7 through GO biological process analysis, GO cellular component analysis, and KEGG analysis of the peroxisomal proteome.

[0045] D. Peroxisomal proteins identified from peroxisomes isolated and purified by the method of Example 7, and known peroxisomal proteins collated according to strict criteria from literature / databases.

[0046] E. GO biological process analysis and KEGG analysis of 208 newly identified peroxisomal proteins in Example 10.

[0047] F. Transfect HeLa cells stably expressing BFP-SKL and COX IV-EGFP with the CYB5R1 YFP plasmid. Then fix the cells and detect BFP, EGFP, and YFP signals under a microscope. Scale bar: 10 μm.

[0048] Figure 5 Peroxisomal proteomics identification in various tissues of mice.

[0049] A. Proteins identified from peroxisomes isolated and purified from the brains, kidneys, and livers of 8-week-old mice by the method of Example 7.

[0050] B. Proportion of proteins identified from peroxisomes isolated and purified from the brains, kidneys, and livers of 8-week-old mice by the method of Example 7 in the peroxisomal protein reference set.

[0051] C. Expression of known peroxisomal proteins in peroxisomes isolated and purified from various tissues of mice by the method of Example 7.

[0052] D. Proteins detected from peroxisomes analyzed and purified from three tissues by the method of Example 7 through GO biological process analysis, GO cellular component analysis, and KEGG analysis of the peroxisomal proteome.

[0053] E. Newly identified peroxisomal proteins in peroxisomes isolated and purified from various tissues of mice by the method of Example 7.

[0054] F. Peroxisomal proteins identified from peroxisomes isolated and purified from various tissues of mice by the method of Example 7, and known peroxisomal proteins collated according to strict criteria from literature / databases.

[0055] G. Known and newly discovered peroxisomal proteins specifically expressed in the brain, kidney, and liver, identified from peroxisomes isolated and purified from various tissues of mice by the method of Example 7. Detailed implementation mode

[0056] The following specific examples illustrate the implementation modes of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0057] Before further describing the specific implementation modes of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific implementation modes described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific implementation modes and not for limiting the protection scope of the present invention; in the specification and claims of the present invention, unless otherwise clearly indicated in the text, the singular forms "a", "an", and "the" include the plural forms.

[0058] When the embodiments give numerical ranges, it should be understood that unless otherwise specified in the present invention, any value between the two endpoints of each numerical range and the two endpoints themselves can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials of the prior art similar to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0059] Principle of the present invention: The amino acids at positions 2 - 90 at the N-terminus of the ATF6α protein can specifically bind to PMP70 and ABCD1 in peroxisomes, and the higher the concentration of the polypeptide, the stronger its specific binding to PMP70 and ABCD1. Based on this, a method for isolating and purifying peroxisomes is provided. The tagged ATF6αN protein, that is, the tag-ATF6αN fusion protein, is coupled to a medium to form a fusion protein-medium complex. When a sample containing peroxisomes flows through the fusion protein-medium complex, the peroxisomes in the sample specifically bind to the ATF6αN protein on the fusion protein-medium complex. Therefore, the peroxisomes bind to the medium, while other components are not bound to the medium and are discarded. Then, the peroxisomes are eluted from the fusion protein-medium complex with an elution buffer to obtain peroxisomes. The obtained peroxisomes are bound with the fusion protein, and the obtained peroxisomes can be used for subsequent experiments such as flow cytometry, electron microscopy, and proteomic analysis.

[0060] The ATF6α in the present invention, namely cyclic adenosine monophosphate-dependent transcription factor ATF-6α, is a protein located on the endoplasmic reticulum membrane. When an endoplasmic reticulum stress response occurs, it transcriptionally regulates the expression of genes related to the unfolded protein stress response.

[0061] In the present invention, the ATF6αN protein should be a natural or artificially synthesized protein containing the ATF6αN protein sequence.

[0062] Furthermore, the ATF6αN protein in the present invention can be derived from mammals or can be artificially prepared. For example, recombinant ATF6αN protein can be produced according to conventional genetic engineering recombination techniques for use in experiments or clinically.

[0063] The peroxisome in the present invention can be a peroxisome in eukaryotes such as animals, plants, and fungi.

[0064] The PMP70 in the present invention, namely peroxisomal membrane protein 70, is an ATP-binding cassette transporter and one of the main components of the peroxisomal membrane. PMP70 is synthesized on free polysomes, inserted into the peroxisomal membrane after translation, and assembled into a dimer or oligomer form on the peroxisomal membrane, participating in the metabolic transport of long-chain acyl-CoA across the peroxisomal membrane.

[0065] The ABCD1 in the present invention is a peroxisomal membrane protein, an ATP-binding cassette transporter, and a protein located on the intracellular membrane. Its main function is to transport long-chain fatty acids from the cytoplasm into the peroxisome for further metabolism.

[0066] The first aspect of the present invention provides a conjugate, which is a conjugate of the ATF6αN protein and the peroxisome.

[0067] The ATF6αN protein binds to the peroxisomal membrane proteins PMP70 and / or ABCD1.

[0068] The ATF6αN protein is selected from any one of the following:

[0069] a) The amino acid sequence of the ATF6αN protein includes SEQ ID No.1;

[0070] b) The ATF6αN protein has homology with a) and has the ability to bind to the peroxisome.

[0071] The SEQ ID No.1 is:

[0072] GEPAGVAGTMESPFSPGLFHRLDEDWDSALFAELGYFTDTDELQLEAANETYENNFDNLDFDLDLMPWESDIWDINNQICTVKDIKAEP。

[0073] The second aspect of the present invention provides the use of ATF6αN protein in the isolation and purification of peroxidase.

[0074] The ATF6αN protein has the ability to bind to the peroxisomal membrane protein PMP70 and / or ABCD1.

[0075] The ATF6αN protein is selected from any of the following:

[0076] a) The amino acid sequence of the ATF6αN protein includes SEQ ID No.1;

[0077] b) The ATF6αN protein has homology with a) and has the ability to bind to peroxisomes.

[0078] The SEQ ID No.1 is:

[0079] GEPAGVAGTMESPFSPGLFHRLDEDWDSALFAELGYFTDTDELQLEAANETYENNFDNLDFDLDLMPWESDIWDINNQICTVKDIKAEP。

[0080] The third aspect of the present invention provides a method for isolating and purifying peroxisomes, the method comprising the following steps:

[0081] (1) The tag-ATF6αN fusion protein binds to the medium to obtain a fusion protein-medium complex, and the tag can bind to the medium;

[0082] (2) Adding the crude peroxisome sample to the fusion protein-medium complex to obtain a fusion protein-medium-peroxisome complex;

[0083] (3) Adding an elution buffer and collecting the eluate to obtain peroxisomes bound to the tag-ATF6αN fusion protein.

[0084] In certain embodiments of the present invention, the medium is an affinity chromatography medium, including but not limited to affinity chromatography media such as agarose resin and magnetic beads commonly used for the expression and purification of eukaryotic and prokaryotic proteins.

[0085] Adding the crude peroxisome sample to the fusion protein-medium complex further includes incubation, and the incubation conditions can be conventional. For example, the incubation conditions are: at 4°C, mixing by inverting up and down, and incubating for 30 min.

[0086] The obtained fusion protein-media-peroxisome complex further includes washing the fusion protein-media-peroxisome complex, and the washing is repeated at least 3 times.

[0087] The fusion protein on the peroxisome has no influence on subsequent experiments such as proteomic analysis, transmission electron microscopy observation, and flow cytometry analysis of peroxisomes.

[0088] The elution buffer described in the present invention can be conventional. For example, the elution buffer can be PBS containing 1-10 mg / ml reduced glutathione. In certain embodiments of the present invention, the elution buffer can be PBS with 5 mg / ml reduced glutathione.

[0089] The method further includes one or more of the following features A to F:

[0090] A. Step (1) includes the following steps:

[0091] Adding the tag-ATF6αN fusion protein to the medium and incubating to obtain a fusion protein-medium complex;

[0092] B. Step (3) further includes:

[0093] (31) Repeatedly adding the elution buffer for elution and combining the eluates;

[0094] (32) Concentrating the peroxisomes bound with the fusion protein;

[0095] C. The method further includes disrupting the cells or tissues in the cell or tissue suspension, centrifuging and collecting the supernatant to obtain a crude peroxisome sample. Preferably, the cells are from animals, plants or fungi;

[0096] D. The method further includes expressing and purifying the tagged ATF6αN protein to obtain the tag-ATF6αN fusion protein;

[0097] E. The tag is selected from GST, His, short peptide tag, MBP;

[0098] F. The medium is selected from glutathione affinity chromatography medium, Ni-NTA affinity chromatography medium, medium containing short peptide tag antibody, MBP affinity chromatography medium.

[0099] In the feature A,

[0100] The concentration, volume, and transfer amount of the medium can all be conventional, and can be adjusted according to the selection of the medium and the actual situation.

[0101] The addition amount of the tag-ATF6αN fusion protein can be confirmed according to the protein loading of the medium. Usually, the tag-ATF6αN fusion protein can be added in an excessive amount.

[0102] The incubation conditions for adding the tag-ATF6αN fusion protein to the medium can be conventional. For example, the incubation conditions are: incubating at 4°C for 30 min.

[0103] The obtained fusion protein-medium complex further includes washing the fusion protein-medium complex with PBS at least 3 times repeatedly.

[0104] In the said Feature B,

[0105] The elution is repeated at least 3 times.

[0106] If higher requirements for peroxisome concentration are needed in subsequent experiments, the peroxisomes bound with the fusion protein can be concentrated.

[0107] The concentration is carried out using a concentrator tube, and the cut-off molecular weight of the concentrator tube is greater than or equal to 300 kDa. Preferably, the cut-off molecular weight of the concentrator tube is 300 kDa.

[0108] In the said Feature C,

[0109] The cell or tissue suspension can be obtained by conventional methods in the prior art, and the selection, dosage and conditions of the reagents can be adjusted according to the actual situation.

[0110] The cells can be cells of animals, plants or fungi, the tissues can be various tissues of various animals, the animals can be rodents, and the tissues can be tissues in organs such as the brain, liver, and kidney.

[0111] The disruption can be carried out by conventional methods in the prior art, such as using a homogenizer for disruption.

[0112] The centrifugation step can select appropriate rotation speed, centrifugation time and number of centrifugations according to the actual situation and the centrifugation effect. For example, centrifuge twice repeatedly, the first time at 800 g for 10 min at 4°C, and the second time at 8000 g for 10 min at 4°C.

[0113] In the said Feature D,

[0114] The method for separating and purifying peroxisomes further includes expressing and purifying the tagged ATF6αN protein to obtain the tag-ATF6αN fusion protein, including the following steps:

[0115] ① Transformation of competent cells;

[0116] ② The transformed strain was cultured in LB liquid medium containing ampicillin until the OD600 reached 0.6 - 0.8;

[0117] ③ Induce the expression of the tagged - ATF6αN fusion protein in the competent cells;

[0118] ④ Lyse the competent cells, centrifuge after lysis and take the supernatant to obtain the cell lysate;

[0119] ⑤ Obtain the tagged - ATF6αN fusion protein through protein purification methods;

[0120] ⑥ Detect and analyze the tagged - ATF6αN fusion protein obtained in ⑤;

[0121] ⑦ Collect and concentrate the tagged - ATF6αN fusion protein.

[0122] Transformation means introducing plasmid DNA into bacteria. The construction of the plasmid is to insert the ATF6αN protein - coding gene into a plasmid vector, and the plasmid vector can be selected from eukaryotic or prokaryotic protein expression vectors with corresponding tags, such as pGEX series vectors like pGEX6P - 1, pGEX6P - 2, and pGEX6P - 3. The plasmid vector contains a tag, and the tag is used to form a tagged fusion protein. The bacteria are Escherichia coli competent cells. In the examples of the present invention, the plasmid vector is the PGEX6P - 1 vector, and the Escherichia coli competent cells are BL21 Escherichia coli competent cells.

[0123] The method of inserting the ATF6αN protein - coding gene into the plasmid vector is a prior art and can be selected and adjusted according to actual needs. For example, homologous recombination technology, Gibson assembly technology, etc. can be selected.

[0124] The transformation method of competent cells is a prior art and can be selected and adjusted according to actual situations. For example, the transformation method of competent cells can be: Place the BL21 Escherichia coli competent cells on ice for 10 minutes until completely melted; Add 50 ng of plasmid to 50 μl of thawed BL21 cells. Gently flick the tube carefully to mix the cells and DNA. Incubate the mixture on ice for 20 minutes; Heat shock at 42 °C for 60 seconds and immediately place on ice for 2 minutes; Add 900 μl of LB to the mixture and shake vigorously at 37 °C for 45 minutes; Heat the LB plate (containing ampicillin antibiotic) to 37 °C; Spread 100 μl of the diluted mixture onto the LB plate and incubate overnight at 37 °C.

[0125] The culture method of culturing the transformed strain in LB liquid medium containing ampicillin until OD600 reaches 0.6 - 0.8 is a prior art and can be selected and adjusted according to actual situations. For example, it can be divided into two-step culture. First, pick a single colony of the expression strain into 20 ml of LB containing ampicillin, shake well at 220 rpm overnight at 37°C; add the 20 ml of bacterial liquid the next day into 1 L of LB containing ampicillin, and incubate for about 4 hours until OD600 reaches 0.6 - 0.8.

[0126] In some embodiments of the present invention, the inducer used to induce the expression of ATF6αN protein in competent cells is IPTG. The concentration of the IPTG used is conventional. Preferably, the concentration of the IPTG is 1 mM. The induction of the expression of ATF6αN protein in competent cells further includes inducing by shaking after adding the inducer.

[0127] The lysis of the competent cells is to resuspend the cells in lysis buffer and lyse the cells by sonication. The lysis of the competent cells is a prior art and can be selected and adjusted according to actual situations. For example, it can be: centrifuge the bacterial liquid at 4000 g for 15 minutes, discard all the supernatant; resuspend the cell pellet in 20 ml of pre-cooled lysis buffer; lyse the cells by sonication; centrifuge the lysis buffer containing cell debris at 4°C and 40000 g for 45 minutes, and transfer the supernatant to a 50 ml tube to obtain cell lysate, thereby realizing the lysis of the competent cells.

[0128] The protein purification method can be selected from techniques such as protein precipitation, buffer exchange, ion exchange chromatography, affinity chromatography, hydrophobic interaction, size exclusion chromatography, electrophoresis, etc. For example, in some embodiments of the present invention, affinity chromatography technology is used to obtain the tag-ATF6αN fusion protein. When the tag is GST, the specific operation can be: resuspend glutathione affinity resin, and transfer 2 ml of the resin (50% suspension) to a 10 ml gravity column; equilibrate the resin with 30 ml of wash buffer, add the cell lysate to the resin, and collect the flow-through; wash the resin three times with 10 ml of wash buffer each time; elute the protein with 2 ml of elution buffer and collect the elution buffer containing the protein; repeat the elution step 4 times; concentrate the protein with a 10 kDa protein concentrator tube, and use a desalting column Sephadex G-25 (Cytiva) to remove the reduced glutathione in the protein eluate.

[0129] The detection and analysis of the tag-ATF6αN fusion protein include analyzing the protein expression by SDS-PAGE to determine the protein concentration in the eluate, and staining the gel with Coomassie Brilliant Blue reagent to facilitate the observation of protein bands.

[0130] Among the said feature E, the short peptide tags are selected from: Myc, HA, V5, Flag, Strep.

[0131] In an embodiment of the present invention, the tag and the medium can be combined.

[0132] In certain embodiments of the present invention, the tag is GST.

[0133] The said GST is glutathione S-transferase, and the GST tag can specifically bind to its substrate glutathione.

[0134] In certain embodiments of the present invention, the medium is glutathione affinity resin.

[0135] Ceapin-A7

[0136] A selective blocker of endoplasmic reticulum stress ATF6α signal, with an IC50 value of 0.59 μM.

[0137] PEX19 KO cells

[0138] PEX19 KO cells are HeLa cells in which PEX19 in HeLa cells has been knocked out by a conventional method, resulting in the absence of PMP70 and ABCD1 in HeLa cells.

[0139] WT cells

[0140] Wild-type HeLa cells.

[0141] SKL

[0142] A tripeptide sequence on the peroxisomal matrix protein. Most peroxisomal matrix proteins carry the peroxisomal targeting sequence 1 (PTS1), which is located at the carboxyl terminus and consists of the tripeptide "SKL" or its variants and nine less conserved amino acids upstream.

[0143] COXIV

[0144] Cytochrome c oxidase subunit IV, which is one of the many subunits of cytochrome c oxidase, is located on the inner mitochondrial membrane and is the terminal enzyme of the mitochondrial respiratory chain.

[0145] SEC61B

[0146] The SEC61 complex is the core component of the endoplasmic reticulum membrane protein transport device, and this complex consists of three membrane proteins α, β, and γ. The oligomer of the SEC61 complex can form a transmembrane channel, through which proteins are transported across the membrane and integrated into the endoplasmic reticulum membrane.

[0147] FLAG

[0148] The FLAG-tag protein is a fusion polypeptide of 8 amino acids (DYKDDDDK). As a fusion expression tag, FLAG usually does not interact with the target protein and generally does not affect the function and properties of the target protein.

[0149] GFP

[0150] That is, green fluorescent protein, which is a protein composed of about 238 amino acids. It can be excited by blue light to ultraviolet light and emit green fluorescence. The green fluorescent protein gene is commonly used as a reporter gene.

[0151] BPF

[0152] BPF is blue fluorescent protein, which contains 259 amino acids. Using the fluorescence characteristics of BFP, it is relatively easy to observe the expression level and intracellular localization of the fusion protein. The fusion protein can also be detected or immunoprecipitated using BFP antibody.

[0153] mCherry

[0154] mCherry is the best monomeric red fluorescent protein evolved from DsRed. It can be used in common with GFP series fluorescent proteins to achieve multicolor labeling. When mCherry fuses foreign proteins at the N-terminus and C-terminus, the fluorescence protein activity and the function of the fused target protein do not significantly affect each other.

[0155] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be described in detail below in conjunction with embodiments. However, they should not be construed as limiting the protection scope of the present invention.

[0156] In the following embodiments, unless otherwise specified, all are conventional methods.

[0157] In the following embodiments, the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0158] Example 1 Molecular Cloning

[0159] The chimeric gene of ABCD1 (chABCD1, synthesized by GENEWIZ) was cloned into the pEG–BacMam plasmid vector with an N-terminal FLAG tag (DYKDDDDK) through Gibson assembly. The PMP70 gene was amplified by PCR and cloned into the pCDNA3.1 plasmid vector with a C-terminal FLAG tag through Gibson assembly. The ATF6 N protein gene was amplified from HeLa cDNA and cloned into the pEGFP-C1 plasmid vector through Gibson assembly and placed between the BamHI and EcoRI sites for the preparation of a fusion protein with GFP. The ATF6 N protein gene was amplified by PCR and cloned into the PGEX6P-1 vector between the BamHI and EcoRI sites for the preparation of the GST-ATF6 N fusion protein. BFP-SKL, COX-GFP, and SEC61b-mCherry were amplified by PCR and cloned into the lentiviral vector phrr-ef1α between the BamHI and NotI sites through Gibson assembly.

[0160] Example 2 Cell Culture

[0161] HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum under the conditions of 37 °C and 5% CO2.

[0162] Escherichia coli BL21 cells were cultured at 37 °C in 1 L of LB medium supplemented with 100 μg / ml ampicillin.

[0163] Example 3 Expression and Purification of Membrane Proteins PMP70 and ABCD1

[0164] HEK293T cells were cultured in 15-cm culture dishes and transfected with 50 μg of the pEG–BacMam plasmid vector and the pCDNA3.1 plasmid vector prepared in Example 1 containing 125 μl of polyethyleneimine (PEI) in serum-free medium at 60% confluence. After 48 h of transfection, the HEK293T cells were centrifuged at 800 g for 10 minutes. The HEK293T cell pellet was resuspended in a lysis buffer containing 25 mM pH 7.5 Tris-HCl, 150 mM NaCl, 20% (v / v) glycerol, 1 mM DTT, 1% (w / v) LMNG (NG310, Anatrace), and 0.1% (w / v) CHS (C6013-25, Anatrace). The cells were disrupted in a 7-ml Dounce homogenizer and then centrifuged at 45000 rpm for 45 minutes, and the supernatant was collected to obtain the cell lysate. The cell lysate was applied to an anti-flag M2 affinity gel (Genscript) and rotated overnight at 4°C. The anti-flag M2 affinity gel was washed with a wash buffer containing 25 mM pH 7.5 Tris-HCl, 150 mM NaCl, 10% (v / v) glycerol, 1 mM DTT, and 0.06% (w / v) digitonin (Beyotime). The protein was eluted with an elution buffer containing 25 mM pH 7.5 Tris-HCl, 150 mM NaCl, 5% (v / v) glycerol, 1 mM DTT, 0.06% (w / v) digitonin, and 200 μg / ml FLAG peptide to obtain the FLAG-tagged membrane proteins Flag-PMP70 and Flag-ABCD1.

[0165] Example 4 Expression and purification of GST-ATF6 N fusion protein.

[0166] Plasmids encoding GST or GST-ATF6 N fusion protein were transformed into Escherichia coli BL21. After transformation, protein expression was induced with 1 mM IPTG at 20°C for 4 hours. The cells were collected by centrifugation, resuspended in a lysis buffer (50 mM pH 7.5 Tris, 150 mM NaCl), lysed by sonication, and centrifuged at 40000 rpm for 40 minutes at 4°C after lysis, and the supernatant was taken to obtain the cell lysate. The cell lysate was purified with equilibrated glutathione affinity resin (Genscript) and washed three times. The GST-tagged ATF6 N protein was eluted with an elution buffer (50 mM pH 7.5 Tris, 150 mM NaCl, 5 mg / ml reduced glutathione). Reduced glutathione in the eluted protein was removed using a Sephadex desalting column Sephadex G-25 (Cytiva) to obtain the GST-ATF6 N fusion protein.

[0167] Example 5 Association between ATF6 N protein and PMP70

[0168] Experimental method:

[0169] Amplify the ATF6 N protein gene from HeLa cDNA, clone it into the pEGFP-C1 plasmid vector by Gibson assembly, and place it between the BamHI and EcoRI sites. Transfect the pEGFP-C1 plasmid vector into HeLa cells for 24 h, treat the HeLa cells with Ceapin-A7 or DMSO, then fix the HeLa cells and perform PMP70 (Alexa Fluor 555) staining and immunofluorescence analysis;

[0170] Amplify the ATF6 N protein gene from HeLa cDNA, clone it into the pEGFP-C1 plasmid vector by Gibson assembly, and place it between the BamHI and EcoRI sites. Transfect HEK293T cells with the pEGFP-C1 plasmid vector for 24 h, and then treat them with 6 μM DMSO or Ceapin-A7 for 24 hours. Lyse the HEK293T cells, immunoprecipitate the cell lysate with a GFP antibody, and perform immunoblotting with the indicated antibody.

[0171] Experimental results:

[0172] As Figure 1 shown in A, immunofluorescence analysis showed that the GFP-ATF6 N protein was expressed in the cytoplasm of DMSO-treated cells and co-localized with PMP70 in Ceapin-A7-treated cells. Then, immunoprecipitate the GFP-ATF6 N protein from the cell lysate containing detergent. As Figure 1 shown in B, there was an interaction between the GFP-ATF6 N protein and PMP70 regardless of the presence of Ceapin-A7.

[0173] Example 6 Binding of GST-ATF6 N fusion protein to PMP70 and ABCD1

[0174] Experimental method:

[0175] Obtain the GST-ATF6 N fusion protein by the expression and purification of the GST-ATF6 N fusion protein in Example 4.

[0176] In vivo pull-down assay: HeLa cells transfected with the pCDNA3.1 plasmid vector and HeLa cells transfected with the pEG–BacMam plasmid vector were lysed separately using HeLa lysis buffer (20 mM Tris at pH 7.5, 150 mM NaCl, 1% Triton X-100, and protease inhibitors). The HeLa cell lysates were centrifuged at 13,000 x g for 10 minutes at 4°C. The GST-ATF6 N fusion protein was added to the centrifuged supernatants separately to obtain a protein mixture of PMP70 and the fusion protein and a protein mixture of ABCD1 and the fusion protein. The two protein mixtures were incubated with glutathione affinity resin at 4°C for 2 hours. Then the glutathione affinity resin was collected separately, washed three times with the wash buffer, and immunoblotting was performed.

[0177] In vitro pull-down assay: Flag-PMP70 and Flag-ABCD1 obtained in Example 3 were mixed with the GST-ATF6N fusion protein separately to obtain two protein mixtures of Flag-PMP70 and the fusion protein and Flag-ABCD1 and the fusion protein, and incubated at 4°C for 30 minutes in a buffer containing 25 mM Tris-HCl at pH 7.5 and 150 mM NaCl. Glutathione affinity resin or anti-flag M2 affinity gel was added to the two protein mixtures separately and incubated at 4°C for 1 hour. Then the glutathione affinity resin or anti-flag M2 affinity gel was collected separately, washed three times with the wash buffer (25 mM Tris-HCl at pH 7.5 and 150 mM NaCl), and SDS-PAGE or immunoblotting was performed.

[0178] Co-immunoprecipitation and immunoblotting: GFP antibody and protein G beads were added to the protein mixtures of Flag-PMP70 and the fusion protein and Flag-ABCD1 and the fusion protein for immunoprecipitation, and incubated with rotation at 4°C overnight. The immune complexes were centrifuged into pellets and washed three times with the lysis buffer. The immune complexes were boiled with SDS loading buffer and heated at 95°C for 10 min. The supernatant was subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, IPVH00010), and probed with a primary antibody and an indicator secondary antibody conjugated with horseradish peroxidase. The signal was detected using an enhanced chemiluminescence substrate (Epizyme) according to the manufacturer's instructions.

[0179] Increasing gradients of GST-ATF6 N protein were incubated with the HeLa cell lysate, then treated with 6 μM DMSO or Ceapin-A7 for 2 hours and the above pull-down assay, co-immunoprecipitation, and immunoblotting experiments were repeated.

[0180] Experimental results:

[0181] In vivo pull-down assay: As shown in Figure 1 Figures D - E, the GST-ATF6 N protein obtained through Example 4 could pull down PMP70 from the HeLa cell lysate, indicating that there was an interaction between the GST-ATF6 N protein and PMP70, and when more GST-ATF6 N protein was added to the cell lysate, the interaction was enhanced. And in the HeLa cell lysate containing a high content of GST-ATF6 N protein, the peroxisomal membrane protein PEX13 was detected in the pull-down complex of the GST-ATF6 N protein( Figure 1 E). As shown in Figure 1 Figure F, immunoblot analysis showed that the GST-ATF6 N protein interacted with both PMP70 and ABCD1.

[0182] In vitro pull-down assay: As shown in Figure 1 Figures G - I, both the purified FLAG-ABCD1 and FLAG-PMP70 proteins could bind to the GST-ATF6 N protein in vitro. As shown in Figure 1 Figures J - K, the GST pull-down assay further showed that the GST-ATF6N protein did not bind to other peroxisomal proteins such as PEX19 and PEX5.

[0183] The above results indicated that the ATF6 N protein could specifically interact with ABCD1 and PMP70 regardless of the presence of Ceapin-A7.

[0184] Example 7 Separation of peroxisomes by GST-ATF6 N protein-binding affinity chromatography

[0185] Experimental method:

[0186] For HeLa cells, the cells were washed 3 times with PBS, resuspended in PBS, disrupted with a 7 ml Dounce homogenizer using Pestle B, and struck 80 times. After disruption with the homogenizer, the mixture was centrifuged at 800 x g at 4 °C for 10 minutes. The supernatant was transferred to a new centrifuge tube and centrifuged at 8000 x g at 4 °C for 10 minutes, and the supernatant was transferred to a new centrifuge tube to obtain a crude peroxisome sample.

[0187] For mouse tissues, the specific tissues were cut into small pieces and washed 3 times with PBS. The tissue pieces were suspended in PBS, disrupted with a 7 ml Dounce homogenizer using Pestle B, and then the centrifugation steps were the same as those for HeLa cells.

[0188] The GST-ATF6 N fusion protein was obtained according to Example 4 for the expression and purification of the GST-ATF6 N fusion protein.

[0189] Resuspend the glutathione affinity resin and transfer 400 μl of the resin (50% suspension) into a 3 ml gravity column; equilibrate the precipitated resin with 10 ml of PBS, add 4 mg of the purified GST-ATF6 N fusion protein to the resin to obtain a fusion protein-resin complex, and incubate at 4°C for 30 minutes; wash the precipitated fusion protein-resin complex 3 times with PBS; add the crude peroxisome sample to the fusion protein-resin complex and incubate at 4°C for 30 minutes to obtain a fusion protein-resin-peroxisome complex; discard the flow-through, wash 3 times with 3 ml of ice-cold PBS; elute the peroxisomes with 200 μl of elution buffer (PBS containing 5 mg / ml reduced glutathione), and collect the eluate; repeat the elution step 4 more times, and combine the 5 elution fractions; the purified peroxisomes can be concentrated through a 100 kDa concentrator tube (Millipore).

[0190] Experimental results:

[0191] The peroxisomes were isolated and purified by the above method. After identification, the obtained peroxisomes were bound to ATF6 N. Examples 8-9 show that the method for isolating and purifying peroxisomes of the present invention can effectively isolate and purify peroxisomes from other cell components.

[0192] Example 8 Purity and efficiency of the peroxisomes isolated and purified by the present invention

[0193] Experimental method:

[0194] A HeLa cell line (CSS cell line) stably expressing BFP-SKL, COXIV-EGFP, and mCherry-SEC61B was constructed to fluorescently label the peroxisome matrix, mitochondrial membrane, and endoplasmic reticulum membrane, respectively. The specific operations are as follows: Culture HEK293T cells, and transfect HEK293T cells with the lentiviral vectors pCMV-dR8.91 and pMD2G, respectively. Harvest the cell culture medium containing lentivirus 48 h after transfection. Filter the virus supernatant with a 0.45 μm filter membrane. Infect HeLa cells with BFP-SKL, COX-EGFP, and SEC61b-mCherry lentiviruses at 50% confluence. Fluorescence-activated cell sorting technology for BFP + , GFP + , mCherry +Triple-positive cells were sorted into 96-well plates, with 1 cell per well for monoclonal selection. The expression and localization of BFP, GFP, and mCherry were examined using a confocal microscope. The peroxisomes were isolated and purified using the method of Example 7. During the isolation and purification process of Example 7, the proportion of the three fluorescence signals in the peroxisomes obtained by the isolation and purification of Example 7 was monitored by immunofluorescence. The abundance of peroxisome-related marker proteins was analyzed by immunoblotting.

[0195] Experimental results:

[0196] As Figure 2 shown in B-C, the proportion of BFP-SKL gradually increased, while the proportions of COXIV-EGFP and mCherry-SEC61B gradually decreased. In the peroxisomes obtained by the isolation and purification of Example 7, almost only the fluorescence of BFP-SKL could be observed, while the fluorescence of COXIV-EGFP and mCherry-SEC61B was almost absent, indicating that the content of the peroxisome matrix obtained by the isolation and purification of Example 7 gradually increased and the purity of the purified peroxisomes was relatively high, with almost no other impurities. As Figure 2 shown in D, the presence of various organelles could be detected in the lysates of WT cells, the presence of PMP70 and ABCD1 could not be detected in the PEX19 KO cells, and peroxisomal membrane proteins and matrix proteins could be detected in the peroxisome samples of WT cells, but not in the peroxisome samples of PEX19 KO cells. In the peroxisome samples isolated and purified in Example 7 from both WT and PEX19 KO cells, almost no other organelle markers were detected.

[0197] Example 9 evaluated the activity of peroxisomes isolated and purified by the method of the present invention

[0198] Experimental method:

[0199] The peroxisomes isolated and purified in Example 7 were lysed in 200 μl of extraction buffer containing 1% Triton X-100 to obtain a lysate. The lysate was centrifuged at 13,000 x g for 10 minutes at 4 °C, and the supernatant was transferred to a new tube. Catalase activity: The supernatant was detected using a CheKine Catalase (CAT) Activity Assay Kit (Abbkine). Glutamate dehydrogenase (GDH) activity: The supernatant was detected using a GDH Activity Assay Kit (Sangon Biotech). Carboxylesterase (CarE) activity: The supernatant was detected using a Carboxylesterase (CarE) Activity Assay Kit (Sangon Biotech). Calculations were performed according to the manufacturer's methods. The enzyme activities were normalized to the protein concentration in the supernatant using the BCA method.

[0200] Experimental results:

[0201] The enzyme activities of peroxisome, mitochondrial and endoplasmic reticulum marker enzymes in the peroxisomes isolated and purified in Example 7 were measured. As Figure 2 shown in E-G, catalase activity was detected only in the peroxisomes of WT cells.

[0202] Example 10 Comparison of the method for isolating and purifying catalase bodies of the present invention with the traditional density gradient centrifugation method

[0203] Experimental method:

[0204] For the isolation of peroxisomes by traditional density gradient centrifugation, a peroxisome isolation kit (PEROX1, Sigma) was used to isolate peroxisomes according to the manufacturer's instructions: HeLa cells were collected and suspended in peroxisome extraction buffer. The cell suspension was transferred to a Dounce homogenizer and homogenized 80 times using Pestle B. The homogenized cell suspension was centrifuged at 1000 x g for 10 minutes at 4 °C. The supernatant was transferred to a new centrifuge tube and centrifuged at 2000 x g for 10 min at 4 °C. The supernatant was centrifuged at 25000 x g for 20 min at 4 °C. The supernatant was discarded and the pellet was resuspended in peroxisome extraction buffer.

[0205] For the density gradient centrifugation step, a gradient mixer was used before use to pour in a linear nykodenz density gradient separation solution ranging from 1.14 to 1.20 g / ml. The pellet suspension was layered on top of the nykodenz gradient and the tube was sealed. Centrifugation was carried out at 100,000 x g for 3 hours at 4 °C. Fractions were collected using a Biocomp system.

[0206] Experimental results: The methods of Example 7 and this example were respectively applied to isolate peroxisomes from the CSS cell line. The results are as Figure 3 shown in A. The peroxisomes isolated and purified by the method of Example 7 contained almost no COXIV-EGFP and SEC61B-mCherry signals, and the BFP-SKL signal was evenly distributed. Three fluorescent signals were detected in the part with the most BFP-SKL signal in the peroxisomes isolated and purified in this example. A certain number of SEC61B-mCherry signals were found to be present in this part, and both the SEC61B-mCherry and BFP-SKL signals showed a clustered distribution. As Figure 3 shown in B, immunoblotting showed that the peroxisomes isolated and purified by density gradient centrifugation in this example were also contaminated by other organelles. However, few components of other organelles were detected in the peroxisomes isolated and purified in Example 7.

[0207] Example 11 Flow Cytometry Analysis of Peroxisomes

[0208] Experimental method: Peroxisomes isolated and purified from WT cells and cells stably expressing EGFP-SKL by the method of Example 7 were compared. The specific method was as follows: The peroxisomes isolated from WT cells or GFP-SKL cells by Example 7 were suspended in PBS, and primary and secondary antibodies were used for incubation to fix WT HeLa cells and GFP-SKL cells. The peroxisomes isolated and purified from WT HeLa cells and GFP-SKL cells were stained with PEX14 antibody (Alexa Fluor 555). After staining, the peroxisomes were eluted and analyzed by CytoFLEX (Beckman Coulter). The data were analyzed using FlowJo version 10 software.

[0209] Experimental results: As Figure 3 shown in C, flow cytometry analysis showed that the EGFP signal of EGFP-SKL peroxisomes isolated and purified by the method of Example 7 was much higher than that of WT peroxisomes. Figure 3 D and E are peroxisomes after staining with PEX14 antibody in flow cytometry.

[0210] Example 12 Transmission Electron Microscopy Observation

[0211] Experimental method: Transmission electron microscopy was used to observe the peroxisomes isolated and purified in Example 7. The specific method was as follows: 4 μL of the peroxisome solution isolated and purified in Example 7 was applied to a Quantifoil R2 / 2 200-mesh holey carbon gold grid (Quantifoil, Micro Tools GmbH, Germany), and luminescence was carried out for 45 s in an H 2 / O 2 atmosphere using Solarus 950 (Gatan, USA). Under the conditions of 4 °C and 90% indoor humidity, it was immediately blotted dry from the back of the grid for 5.0 s, the horizontal blotting position was 44 mm, and the vertical blotting position was 2.5 mm. Then the grid was immersed in liquid ethane-methane using a Leica EM GP2 automatic blotting freezer (Leica Microsystems GmbH, Austria).

[0212] The grid was imaged using a 300 keV Titan Krios transmission electron microscope (Thermo Fisher Scientific, USA) and a K3 direct electron detector (Gatan, USA). Images were acquired using SerialEM software, and the nominal magnification was 29,000× (pixel size ), with a defocus of 2.5 μm and a total dose of

[0213] Experimental results: As Figure 3 shown in F, the cryo - TEM (cryo - transmission electron microscopy) analysis results showed the high resolution of the peroxisomes isolated and purified in Example 7, showing the integrity of the peroxisome membrane and the protein core in the peroxisomes isolated and purified in Example 7, proving that the method for isolating and purifying peroxisomes of the present invention is effective.

[0214] Example 13 Analysis of the Peroxisome Proteome of Mammalian WT HeLa Cells

[0215] Experimental method: A peroxisome protein reference set containing 103 peroxisome proteins was obtained through literature / database (UniProt). The peroxisome protein reference set was identified by mass spectrometry analysis. Label - free quantitative mass spectrometry analysis was used to analyze the peroxisomes isolated and purified from mammalian WT HeLa cells and PEX19 KO cells in Example 7. Among them, the purified peroxisomes in PEX19 KO cells were used as a control precipitate.

[0216] Experimental results: The peroxisomes isolated and purified from mammalian WT HeLa cells and PEX19 KO cells in Example 7 are as Figure 4 shown in A. As Figure 4 shown in B and D, a total of 280 peroxisome proteins were identified from mammalian WT HeLa cells, among which 72 belonged to the above - mentioned peroxisome protein reference set and 208 were new peroxisome candidate proteins. As Figure 4 shown in C, through gene ontology (GO) enrichment analysis and KEGG pathway analysis, the 280 peroxisome proteins have functions related to peroxisomes and functions related to peroxisome isomers, such as fatty acid metabolic processes and lipid oxidation. As Figure 4 shown in E, a total of 208 peroxisome proteins are new peroxisome candidate proteins, which are enriched in fatty acid metabolic processes and glycosylation processes and are related to fatty acid metabolic processes and glycosylation processes. As Figure 4 shown in F, among the 208 new peroxisome candidate proteins, CTP1A and AKAP1 are localized in mitochondria, and CYB5R1 is localized in the endoplasmic reticulum. The YFP - labeled candidate was transfected into HeLa cells, and it was observed that the CYB5R1 signal co - localized with mitochondria and peroxisomes. This example illustrates that the peroxisomes isolated and purified in Example 7 can be used for subsequent proteomic analysis.

[0217] Example 14 Tissue - Specific Analysis of the Peroxisome Proteome of Mice

[0218] Experimental method: Peroxisomes were isolated and purified from the brains, livers, and kidneys of mice by the method of Example 7. To study the heterogeneity of peroxisomal proteins in different tissues of mice, a label-free proteomic analysis was developed to analyze and compare the peroxisomal proteomes isolated and purified from the brains, livers, and kidneys (n = 3) of mice.

[0219] As Figure 5 Shown in A - B, in mass spectrometry analysis, 3907 peroxisomal proteins were identified in brain peroxisomes, accounting for 70.9% of the reference peroxisomal proteins; 3649 peroxisomal proteins were identified in liver peroxisomes, accounting for 89.3% of the reference peroxisomal proteins; 5321 peroxisomal proteins were identified in kidney peroxisomes, accounting for 94.2% of the reference peroxisomal proteins.

[0220] As Figure 5 Shown in A, 2333 peroxisomal proteins were common to the three tissues. As Figure 5 Shown in C, the expression of most peroxisomal proteins showed no significant difference among the three tissues. As Figure 5 Shown in C and G, some peroxisomal proteins showed tissue specificity. For example, Syt7 was only found in the brain, Mpv17l was only found in the kidney, Hao1 and Urad were only found in the liver; some proteins were not detected in some tissues. For example, Tmem35a and Dao were not detected in the liver. Pipox, Slc25a17, Pex11g, Pxmp4, Acot4, Pxmp2, Pex11a, Pex7, Crat, Xdh, Idi1, Agxt2, and Plaat3 were absent in the brain. Figure 5 Shown in D, KEGG pathway and GO Term Process analysis confirmed the enrichment of peroxisome-related processes. Figure 5 Shown in C and 5G, some proteins were not detected in some tissues. Tmem35a and Dao were not detected in the liver. Pipox, Slc25a17, Pex11g, Pxmp4, Acot4, Pxmp2, Pex11a, Pex7, Crat, Xdh, Idi1, Agxt2, and Plaat3 were not detected in the brain. This indicates that the peroxisomes isolated and purified by Example 7 can be used for subsequent proteomic analysis.

[0221] Data statistics of Example 15

[0222] Quantitative data are expressed as mean ± standard deviation, and the significance of the difference between two groups was evaluated using the P value calculated by unpaired two-tailed t-test in GraphPad Prism.

[0223] When an embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the examples, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

Claims

1. A conjugate, which is a conjugate of ATF6αN protein and peroxisome.

2. The conjugate according to claim 1, wherein, the ATF6αN protein binds to the peroxisomal membrane proteins PMP70 and / or ABCD1.

3. The conjugate according to claim 1, wherein, the ATF6αN protein is selected from any one of the following: a) the amino acid sequence of the ATF6αN protein includes SEQ ID No.1; b) the ATF6αN protein has homology with a) and has the ability to bind to peroxisome.

4. Use of ATF6αN protein in the isolation and purification of peroxisome.

5. The use according to claim 4, wherein, the ATF6αN protein has the ability to bind to the peroxisomal membrane proteins PMP70 and / or ABCD1.

6. The use according to claim 4, wherein, the ATF6αN protein is selected from any one of the following: a) the amino acid sequence of the ATF6αN protein includes SEQ ID No.1; b) the ATF6αN protein has homology with a) and has the ability to bind to peroxisome.

7. A method for isolating and purifying peroxisome, wherein, the method comprises the following steps: (1) The tag-ATF6αN fusion protein binds to the medium to obtain a fusion protein-medium complex, and the tag can bind to the medium; (2) Adding a crude peroxisome sample to the fusion protein-medium complex to obtain a fusion protein-medium-peroxisome complex; (3) Adding an elution buffer and collecting the eluate to obtain peroxisome bound with the tag-ATF6αN fusion protein.

8. According to the method of claim 7, wherein, it further comprises one or more of the following features A to F: A. The step (1) includes the following steps: Co-incubating the tag-ATF6αN fusion protein with the medium to obtain a fusion protein-medium complex; B. The step (3) further includes: (31) Repeatedly adding the elution buffer for elution and combining the eluates; (32) Concentrating the peroxisome bound with the fusion protein; C. The method further includes disrupting the cells or tissues in the cell or tissue suspension, centrifuging and collecting the supernatant to obtain a crude peroxisome sample. Preferably, the cells are from animals, plants or fungi; D. The method further includes expressing and purifying the tagged ATF6αN protein to obtain the tag-ATF6αN fusion protein; E. The tag is selected from GST, His, short peptide tag, MBP; F. The medium is selected from glutathione affinity chromatography medium, Ni-NTA affinity chromatography medium, medium containing short peptide tag antibody, MBP affinity chromatography medium.

9. According to the method of claim 7, wherein, the ATF6αN protein in the tag-ATF6αN fusion protein can specifically bind to the peroxisomal membrane proteins PMP70 and / or ABCD1.

10. According to the method of claim 7, wherein, The ATF6αN protein is selected from any of the following: a) The amino acid sequence of the ATF6αN protein comprises SEQ ID No. 1; b) The ATF6αN protein has homology with a) and has the ability to bind to peroxisomes.

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