Method for isolating and purifying peroxisomes

Abstract

Provided are a conjugate of the protein ATF6αN and peroxisomes, a use of the protein ATF6αN in the isolation and purification of peroxisomes, and a method for isolating and purifying peroxisomes by using the protein ATF6αN. The method comprises the following steps: combining a tag-ATF6αN fusion protein with a medium, so as to obtain a fusion protein-medium complex; adding a crude peroxisome sample to the fusion protein-medium complex, so as to obtain a fusion protein-medium-peroxisome complex; and adding an elution buffer and collecting an eluent, so as to obtain peroxisomes conjugated with a fusion protein.

Description

A method for separating and purifying peroxisomes Technical Field

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

[0002] Cells contain a variety of organelles, which interact with each other both functionally and physically. Peroxisomes are tiny organelles in the cytoplasm, approximately 0.1-1 μm in diameter. They are crucial sites for key metabolic activities such as fatty acid α- and β-oxidation and oxidative stress. Peroxisomes have been reported to interact with organelles such as mitochondria, the endoplasmic reticulum, and lysosomes.

[0003] Peroxisomes are closely linked to the development of various diseases, including tumorigenesis, tumor sensitivity to chemotherapeutic drugs, antiviral infections, and Alzheimer's disease. The simple and easy peroxisome isolation technology not only provides a technical means for studying the pathogenesis of various diseases, but also facilitates the development of tumor-targeted drugs targeting peroxisomes and the development of early diagnostic indicators for diseases such as tumors and Alzheimer's.

[0004] Current technologies for isolating and purifying peroxisomes include density gradient centrifugation, immunoprecipitation targeting the peroxisomal membrane protein PMP70, and free-flow electrophoresis. For example, Manner A, Islinger M. Isolation of Mammalian Peroxisomes by Density Gradient Centrifugation. Methods Mol Biol, 2023, 2643: 1-12. disclose a scheme for purifying peroxisomes from liver tissue or HepG2 liver cancer cell lines using density gradient centrifugation. However, existing technologies for isolating and purifying peroxisomes may have problems such as cumbersome methods, high requirements for instrumentation, insufficient purity, the isolated peroxisomes are usually suspended in the gradient solution, which is not conducive to subsequent experiments, and high costs.

[0005] Summary of the Invention

[0006] In view of the above shortcomings of the prior art, the first aspect of the present invention provides a conjugate, which is a conjugate of ATF6αN protein and peroxisome.

[0007] The second aspect of the present invention provides use of ATF6αN protein in separating and purifying peroxisomes.

[0008] A third aspect of the present invention provides a method for separating and purifying peroxisomes, the method comprising the following steps:

[0009] (1) the tag-ATF6αN fusion protein binds to the medium to obtain a fusion protein-medium complex, wherein the tag can bind to the medium;

[0010] (2) adding the crude peroxisome sample to the fusion protein-mediator complex to obtain a fusion protein-mediator-peroxisome complex;

[0011] (3) Add elution buffer and collect the eluate to obtain peroxisomes bound to the tag-ATF6αN fusion protein.

[0012] The beneficial effects of the present invention are:

[0013] The separation and purification method provided by the present invention can easily and efficiently isolate relatively pure peroxisomes. This method is simple to operate and does not require complex instrumentation. It can isolate peroxisomes from any cell or tissue, resulting in high-purity peroxisomes suitable for subsequent routine experiments such as protein profiling and flow cytometry. The separation and purification method provided by the present invention has broad application prospects in the study of peroxisomal function and homeostasis, as well as in the study of inter-organelle interactions in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1. Interaction of ATF6αN protein with peroxisomal membrane proteins.

[0015] A. HeLa cells were transfected with a plasmid expressing GFP-ATF6αN protein for 24 h and then treated with DMSO or Ceapin-A7 (6 μM) for 24 h. The cells were then fixed and stained for PMP70 (Alexa Fluor 555). Scale bar: 5 μm.

[0016] B. HEK293T cells were transfected with a plasmid expressing GFP-ATF6αN protein for 24 h and then treated with DMSO or Ceapin-A7 (6 μM) for 24 h. Cell lysates were immunoprecipitated with a GFP antibody and immunoblotted with the indicated antibodies.

[0017] C. Coomassie-stained SDS-PAGE gel showing the expression of GST and GST-ATF6αN proteins.

[0018] D. HeLa cell lysate was incubated with purified GST or GST-ATF6αN protein, and then treated with DMSO or Ceapin-A7 (6 μM) for 2 hours. GST-tagged proteins were captured using glutathione affinity resin, and immunoblot analysis showed that there was an interaction between GST-ATF6αN protein and PMP70, which was independent of the presence of Ceapin-A7.

[0019] E. HeLa cell lysate was incubated with increasing amounts of GST-ATF6αN protein, followed by treatment with DMSO or Ceapin-A7 (6 μM) for 2 hours. Immunoblotting analysis showed that the interaction between GST-ATF6αN and PMP70 increased with the addition of increasing amounts of GST-ATF6αN protein to the cell lysate.

[0020] F. HeLa cell lysate was incubated with purified GST or GST-ATF6αN protein, and the GST-tagged protein was captured using glutathione affinity resin. Immunoblotting analysis showed that GFP-ATF6αN protein interacted with both PMP70 and ABCD1.

[0021] G. The purified PMP70-Flag and GST-ATF6αN proteins were mixed, pulled down with Flag affinity gel, and then Flag and GST were separated by IB.

[0022] H. Purified Flag-ABCD1 and GST-ATF6αN proteins were mixed and pulled down using glutathione affinity resin, and then Flag and GST were pulled down using IB.

[0023] I. Coomassie-stained SDS-PAGE gel showing the interaction between Flag-ABCD1 and GST-ATF6αN proteins at amino acids 2-90.

[0024] JK.GST pull-down experiments showed that GST-ATF6αN protein interacted with neither PEX19 nor PEX5.

[0025] Figure 2. Design and validation of the peroxisome isolation strategy.

[0026] A. Schematic diagram of the peroxisome isolation workflow of the present invention.

[0027] B. Confocal microscopy analysis of CSS cells, cell lysates, crude peroxisomes, and pure peroxisomes isolated from CSS cells using the method of Example 7. Scale bar for CSS is 2 μm. Scale bars for cell lysates, crude peroxisomes, and pure peroxisomes are 10 μm.

[0028] Quantification of fluorescence signals in CB. Values ​​are expressed as mean ± SD, n = 5.

[0029] D. Immunoblot analysis was performed to evaluate the expression of organelle markers in peroxisomes isolated and purified by the method of Example 7 from WT and PEX19 KO cell lysates.

[0030] EG. 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 using the method of Example 7.

[0031] Figure 3. Comparison with traditional density gradient centrifugation.

[0032] A. Representative fluorescence images of crude peroxisomes, peroxisomes isolated and purified in Example 7, and peroxisomes purified by density gradient centrifugation. Scale bar: 20 μm.

[0033] B. Immunoblotting analysis of different organelles obtained by traditional density gradient centrifugation purification and separation and purification in Example 7.

[0034] C. Flow cytometric analysis of peroxisomes isolated and purified from WT and GFP-SKL cells in Example 7.

[0035] 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.

[0036] 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.

[0037] F. Cryo-transmission electron microscopy shows the morphology of peroxisomes isolated and purified by the method of Example 7.

[0038] Fig. 4 Peroxisome proteomic identification of HeLa cells.

[0039] A. Ratio intensity plot of proteins isolated and purified by the method of Example 7 and proteomic quantification of oxidosomes and whole cell lysates.

[0040] B. Quantitative protein volcano plot in the proteomics of peroxisomes isolated and purified from WT HeLa cells and PEX19 KO HeLa cells using the method of Example 7.

[0041] C. 280 peroxisomal proteins were identified from the peroxisomal proteins isolated and purified from WT HeLa cells and PEX19 KO HeLa cells using the method of Example 7 through GO biological process analysis, GO cellular component analysis, and KEGG analysis of the peroxisomal proteome.

[0042] D. Peroxisomal proteins identified from the peroxisomes separated and purified by the method of Example 7, and known peroxisomal proteins sorted from literature / databases according to strict standards.

[0043] E. GO biological process analysis and KEGG analysis of the 208 new peroxisomal proteins identified in Example 10.

[0044] F. HeLa cells stably expressing BFP-SKL and COX IV-EGFP were transfected with the CYB5R1YFP plasmid. The cells were then fixed and the BFP, EGFP, and YFP signals were examined under a microscope. Scale bar: 10 μm.

[0045] Fig. 5. Proteomic identification of peroxisomes in various mouse tissues.

[0046] A. Proteins identified in peroxisomes isolated and purified from the brain, kidney, and liver of 8-week-old mice using the method of Example 7.

[0047] B. The proportion of proteins identified in the peroxisomes isolated and purified from the brain, kidney and liver of 8-week-old mice using the method of Example 7 in the peroxisomal protein reference set.

[0048] C. Expression of known peroxisomal proteins in peroxisomes isolated and purified from various mouse tissues using the method of Example 7.

[0049] D. GO biological process analysis, GO cellular component analysis, and KEGG analysis of peroxisomal proteome The proteins detected in the peroxisomes purified from the three tissues were analyzed using the method of Example 7.

[0050] E. New peroxisomal proteins identified from peroxisomes isolated and purified from various mouse tissues using the method of Example 7.

[0051] F. Peroxisomal proteins identified from peroxisomes isolated and purified from various mouse tissues by the method of Example 7, and known peroxisomal proteins compiled from literature / databases according to strict standards.

[0052] G. Known and newly discovered peroxisomal proteins specifically expressed in the brain, kidney and liver identified from the peroxisomes isolated and purified from various mouse tissues by the method of Example 7. DETAILED DESCRIPTION

[0053] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the contents disclosed in this specification. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0054] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0055] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0056] The principle of the present invention is that amino acids 2-90 at the N-terminus of the ATF6α protein can specifically bind to PMP70 and ABCD1 in peroxisomes, and the higher the polypeptide concentration, the stronger its specific binding to PMP70 and ABCD1. Based on this, a method for isolating and purifying peroxisomes is provided, in which a tagged ATF6αN protein, i.e., a 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, thereby binding the peroxisomes to the medium, while other components are not bound to the medium and are discarded. The peroxisomes are then eluted from the fusion protein-medium complex with an elution buffer to obtain peroxisomes. The obtained peroxisomes are bound to the fusion protein and can be used for subsequent experiments such as flow cytometry, electron microscopy, and protein spectrum analysis.

[0057] The ATF6α described in the present invention, namely the cyclic AMP-dependent transcription factor ATF-6α, is a protein located on the endoplasmic reticulum membrane. When the endoplasmic reticulum stress response occurs, it transcriptionally regulates the expression of genes related to the unfolded protein stress response.

[0058] In the present invention, the ATF6αN protein should be a natural or artificially synthesized protein containing the ATF6αN protein sequence.

[0059] Furthermore, the ATF6αN protein of the present invention may be derived from mammals or may be artificially prepared. For example, recombinant ATF6αN protein may be produced according to conventional genetic engineering recombination techniques for application in experiments or clinics.

[0060] The peroxisome described in the present invention may be a peroxisome in eukaryotic organisms such as animals, plants, and fungi.

[0061] PMP70, or peroxisomal membrane protein 70, described herein, is an ATP-binding cassette transporter and a major component of the peroxisomal membrane. PMP70 is synthesized on free polysomes and post-translationally inserted into the peroxisomal membrane, where it assembles into dimers or oligomers, participating in the metabolic transport of long-chain acyl-CoA across the peroxisomal membrane.

[0062] The ABCD1 described 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.

[0063] The first aspect of the present invention provides a conjugate, which is a conjugate of ATF6αN protein and peroxisome.

[0064] The ATF6αN protein is combined with peroxisomal membrane proteins PMP70 and / or ABCD1.

[0065] The ATF6αN protein is selected from any one of the following:

[0066] a) the amino acid sequence of the ATF6αN protein includes SEQ ID No. 1;

[0067] b) The ATF6αN protein is homologous to a) and has the ability to bind to peroxisomes.

[0068] The SEQ ID No. 1 is:

[0069] The second aspect of the present invention provides use of ATF6αN protein in separating and purifying peroxidase.

[0070] The ATF6αN protein has the ability to bind to peroxisomal membrane proteins PMP70 and / or ABCD1.

[0071] The ATF6αN protein is selected from any one of the following:

[0072] a) the amino acid sequence of the ATF6αN protein includes SEQ ID No. 1;

[0073] b) The ATF6αN protein is homologous to a) and has the ability to bind to peroxisomes.

[0074] The SEQ ID No. 1 is:

[0075] A third aspect of the present invention provides a method for separating and purifying peroxisomes, the method comprising the following steps:

[0076] (1) the tag-ATF6αN fusion protein binds to the medium to obtain a fusion protein-medium complex, wherein the tag can bind to the medium;

[0077] (2) adding the crude peroxisome sample to the fusion protein-mediator complex to obtain a fusion protein-mediator-peroxisome complex;

[0078] (3) Add elution buffer and collect the eluate to obtain peroxisomes bound to the tag-ATF6αN fusion protein.

[0079] In certain embodiments of the present invention, the medium is an affinity chromatography medium, including but not limited to agarose resin, magnetic beads and other affinity chromatography media commonly used for expression and purification of eukaryotic and prokaryotic proteins.

[0080] The crude peroxisome sample is added to the fusion protein-medium complex and incubated. The incubation conditions can be conventional, for example, the incubation conditions are: inverting and mixing at 4° C., and incubating for 30 minutes.

[0081] The step of obtaining the fusion protein-mediator-peroxisome complex further comprises washing the fusion protein-mediator-peroxisome complex, wherein the washing is repeated at least three times.

[0082] The fusion protein on the peroxisome had no effect on subsequent peroxisome protein spectrum analysis, transmission electron microscopy observation, flow cytometry analysis and other experiments.

[0083] The elution buffer of 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 containing 5 mg / ml reduced glutathione.

[0084] The method further includes one or more of the following features:

[0085] A. The step (1) includes the following steps:

[0086] The tag-ATF6αN fusion protein was added to the medium and incubated to obtain a fusion protein-medium complex;

[0087] B. The step (3) further includes:

[0088] (31) Repeatedly add elution buffer and combine the eluates;

[0089] (32) Concentration of peroxisomes bound to the fusion protein;

[0090] C. The method further comprises disrupting the cells or tissues in the cell or tissue suspension, collecting the supernatant after centrifugation to obtain a crude peroxisome sample, preferably, the cells are derived from animals, plants or fungi;

[0091] D. The method further comprises expressing and purifying the tagged ATF6αN protein to obtain a tag-ATF6αN fusion protein;

[0092] E. The tag is selected from GST, His, a short peptide tag, and MBP;

[0093] F. The medium is selected from glutathione affinity chromatography medium, Ni-NTA affinity chromatography medium, medium containing short peptide tag antibody, and MBP affinity chromatography medium.

[0094] In the feature A,

[0095] The concentration, volume and transfer amount of the medium can all be conventional and can be adjusted according to the choice of medium and actual conditions.

[0096] The amount of tag-ATF6αN fusion protein added can be determined based on the protein loading capacity of the medium. Typically, the tag-ATF6αN fusion protein can be added in an excess amount.

[0097] The tag-ATF6αN fusion protein is added to the medium and incubated under conventional conditions, for example, the incubation condition is: incubation at 4° C. for 30 minutes.

[0098] The step of obtaining the fusion protein-medium complex further comprises washing the fusion protein-medium complex with PBS at least three times.

[0099] In the feature B,

[0100] The elution was repeated at least 3 times.

[0101] If subsequent experiments require a higher peroxisome concentration, peroxisomes bound to the fusion protein can be concentrated.

[0102] The concentration is performed using a concentrator tube, and the molecular weight cutoff of the concentrator tube is greater than or equal to 300 kDa. Preferably, the molecular weight cutoff of the concentrator tube is 300 kDa.

[0103] In the feature C,

[0104] The cell or tissue suspension can be obtained by conventional methods in the prior art, and the selection of reagents, dosage and conditions can be adjusted according to actual conditions.

[0105] The cells may be cells of animals, plants or fungi, the tissues may be various tissues of various animals, the animals may be rodents, and the tissues may be tissues in organs such as the brain, liver, and kidney.

[0106] The crushing can be performed using conventional methods in the prior art, such as using a homogenizer.

[0107] The centrifugation step shown can select an appropriate speed, centrifugation time and number of centrifugations according to the actual situation and the centrifugal effect. For example, the centrifugation can be repeated twice, the first time at 800g, centrifugation at 4°C for 10 minutes, and the second time at 8000g at 4°C for 10 minutes.

[0108] In the feature D,

[0109] The method for isolating and purifying peroxisomes also includes expressing and purifying a tagged ATF6αN protein to obtain a tag-ATF6αN fusion protein, comprising the following steps:

[0110] ① Transformation of competent cells;

[0111] ② The transformed strain was cultured in LB liquid medium containing ampicillin until the OD600 reached 0.6-0.8;

[0112] ③ Induce the expression of tag-ATF6αN fusion protein in competent cells;

[0113] ④ Lyse the competent cells, centrifuge after lysis and take the supernatant to obtain cell lysate;

[0114] ⑤ Obtain tag-ATF6αN fusion protein by protein purification method;

[0115] ⑥ Detecting and analyzing the tag-ATF6αN fusion protein obtained in ⑤;

[0116] ⑦ Collect the tag-ATF6αN fusion protein and concentrate it.

[0117] Transformation is the introduction of plasmid DNA into bacteria. The construction of the plasmid is to insert the ATF6αN protein encoding gene into a plasmid vector. The plasmid vector can be selected from a eukaryotic or prokaryotic protein expression vector with a corresponding tag, such as pGEX6P-1, pGEX6P-2 and pGEX6P-3 and other pGEX series vectors. The plasmid vector contains a tag, and the tag is used to form a fusion protein with a tag. The bacteria are Escherichia coli competent cells. In an embodiment of the present invention, the plasmid vector is a PGEX6P-1 vector, and the Escherichia coli competent cells are BL21 Escherichia coli competent cells.

[0118] The method of inserting the ATF6αN protein encoding gene into a plasmid vector is an existing technology and can be selected and adjusted according to the needs of actual conditions. For example, homologous recombination technology, Gibson assembly technology, etc. can be selected.

[0119] Transformation methods for competent cells are currently available and can be selected and adjusted based on actual conditions. For example, the following method can be used: Place BL21 E. coli competent cells on ice for 10 minutes until completely thawed; add 50 ng of plasmid to 50 μl of thawed BL21 cells. Carefully flick the tube to mix the cells and DNA. Incubate the mixture on ice for 20 minutes; heat shock at 42°C for 60 seconds, then 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 an LB plate (containing ampicillin antibiotic) to 37°C; spread 100 μl of the diluted mixture onto the LB plate and incubate at 37°C overnight.

[0120] The method of culturing the transformed strain in LB liquid medium containing ampicillin to an OD600 of 0.6-0.8 is a conventional method and can be selected and adjusted according to actual conditions. For example, the culture can be divided into two steps: first, a single colony of the expression strain is picked and placed in 20 ml of LB containing ampicillin, and shaken at 220 rpm at 37°C overnight; the next day, 20 ml of the bacterial liquid is added to 1 L of LB containing ampicillin and incubated for about 4 hours until the OD600 reaches 0.6-0.8.

[0121] In certain embodiments of the present invention, the inducer used to induce the expression of ATF6αN protein in competent cells is IPTG, and the concentration of IPTG is conventional. Preferably, the concentration of IPTG is 1 mM. The expression of ATF6αN protein in the inducing competent cells also includes induction by shaking after the addition of the inducer.

[0122] The competent cell lysis is performed by resuspending the cells in a lysis buffer and lysing the cells by ultrasonication. The competent cell lysis method is a conventional technique and can be selected and adjusted according to actual conditions. For example, the method can be as follows: centrifuging the bacterial solution at 4000 g for 15 minutes and discarding all the supernatant; resuspending the cell pellet in 20 ml of pre-cooled lysis buffer; lysing the cells by ultrasonication; centrifuging the lysis buffer containing the cell fragments at 40,000 g for 45 minutes at 4°C, and transferring the supernatant to a 50 ml tube to obtain a cell lysate, thereby achieving lysis of the competent cells.

[0123] The protein purification method can be selected from protein precipitation, buffer replacement, ion exchange chromatography, affinity chromatography, hydrophobic interaction, size exclusion chromatography, electrophoresis and other technologies. For example, in certain embodiments of the present invention, affinity chromatography is used to obtain the tag

[0124] -ATF6αN fusion protein. When the tag is GST, the specific steps can be as follows: resuspend the glutathione affinity resin and transfer 2 ml of 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; rinse 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 four more times; concentrate the protein using a 10 kDa protein concentrator tube, and remove the reduced glutathione in the protein eluate using a Sephadex G-25 desalting column (Cytiva).

[0125] Detection and analysis of the tag-ATF6αN fusion protein included analyzing protein expression by SDS-PAGE to determine the protein concentration in the eluate and staining the gel with Coomassie Brilliant Blue reagent to visualize the protein bands.

[0126] In the feature E, the short peptide tag is selected from: Myc, HA, V5, Flag, and Strep.

[0127] In an embodiment of the present invention, the label and the medium may be combined.

[0128] In certain embodiments of the present invention, the tag is GST.

[0129] The GST is glutathione S-transferase, and the GST tag can specifically bind to its substrate glutathione.

[0130] In certain embodiments of the present invention, the medium is a glutathione affinity resin.

[0131] Ceapin-A7

[0132] A selective blocker of ER stress-induced ATF6α signaling with IC50 of 0.59 μM.

[0133] PEX19 KO cells

[0134] PEX19 KO cells are HeLa cells in which PEX19 is knocked out by conventional methods, resulting in the loss of PMP70 and ABCD1 in HeLa cells.

[0135] WT cells

[0136] Wild-type HeLa cells.

[0137] SKL

[0138] Tripeptide sequences on peroxisomal matrix proteins. Most peroxisomal matrix proteins carry a peroxisome 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.

[0139] COXIV

[0140] Cytochrome c oxidase IV is one of the many subunits of cytochrome c oxidase. It is located on the inner membrane of mitochondria and is the terminal enzyme of the mitochondrial respiratory chain.

[0141] SEC61B

[0142] The SEC61 complex is a core component of the endoplasmic reticulum membrane protein transport apparatus. The complex is composed of three membrane proteins, α, β, and γ. Oligomers of the SEC61 complex form a transmembrane channel, through which proteins are transported across the membrane and integrated into the endoplasmic reticulum membrane.

[0143] FLAG

[0144] The FLAG tag protein is an 8-amino acid fusion polypeptide (DYKDDDDK). As a fusion expression tag, FLAG generally does not interact with the target protein and generally does not affect the function or properties of the target protein.

[0145] GFP

[0146] Green fluorescent protein is a protein composed of approximately 238 amino acids that can be excited by light from blue to ultraviolet, emitting green fluorescence. The green fluorescent protein gene is often used as a reporter gene.

[0147] BPF

[0148] BPF is a blue fluorescent protein containing 259 amino acids. The fluorescent properties of BFP can be used to easily observe the expression level and intracellular localization of the fusion protein. BFP antibodies can also be used to detect or immunoprecipitate the fusion protein.

[0149] mCherry

[0150] mCherry is a monomeric red fluorescent protein with the best performance evolved from DsRed. It can be used together with the GFP series of fluorescent proteins to achieve multi-color labeling. When mCherry is fused to exogenous proteins at the N-terminus and C-terminus, the activity of the fluorescent protein and the function of the fused target protein have no obvious effect on each other.

[0151] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0152] In the following examples, unless otherwise specified, all methods are conventional.

[0153] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0154] Example 1 Molecular cloning

[0155] The ABCD1 chimeric gene (chABCD1, synthesized by GENEWIZ) was cloned into the pEG–BacMam plasmid vector with an N-terminal FLAG tag (DYKDDDDK) by Gibson assembly. The PMP70 gene was PCR amplified and cloned into the pCDNA3.1 plasmid vector with a C-terminal FLAG tag by Gibson assembly. The ATF6 N protein gene was amplified from HeLa cDNA and cloned into the pEGFP-C1 plasmid vector between the BamHI and EcoRI sites by Gibson assembly for the production of a GFP fusion protein. The ATF6 N protein gene was PCR amplified and cloned into the pGEX6P-1 vector between the BamHI and EcoRI sites by Gibson assembly for the production of a GST-ATF6 N fusion protein. BFP-SKL, COX-GFP, and SEC61b-mCherry were PCR amplified and cloned into the lentiviral vector phrr-EF1α between the BamHI and NotI sites by Gibson assembly.

[0156] Example 2 Cell culture

[0157] HEK293T cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0158] Escherichia coli BL21 cells were cultured in 1 L of LB medium supplemented with 100 μg / ml ampicillin at 37°C.

[0159] Example 3 Expression and purification of membrane proteins PMP70 and ABCD1

[0160] HEK293T cells were cultured in 15 cm culture dishes and transfected with 50 μg of the pEG–BacMam plasmid vector prepared in Example 1 and the pCDNA3.1 plasmid vector in serum-free medium at 60% confluency. 48 hours after transfection, the HEK293T cells were centrifuged at 800 g for 10 minutes. The HEK293T cell pellet was resuspended in lysis buffer containing 25 mM Tris-HCl (pH 7.5), 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 45,000 rpm for 45 minutes. The supernatant was collected to obtain the cell lysate. Cell lysates were applied to 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 Tris-HCl (pH 7.5), 150 mM NaCl, 10% (v / v) glycerol, 1 mM DTT, and 0.06% (w / v) digitonin (Beyotime). Proteins were eluted with an elution buffer containing 25 mM Tris-HCl (pH 7.5), 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.

[0161] Example 4 Expression and purification of GST-ATF6 N fusion protein.

[0162] Plasmids encoding GST or GST-ATF6 N fusion proteins were transformed into Escherichia coli BL21. Protein expression was induced with 1 mM IPTG at 20°C for 4 hours. Cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl), and lysed by sonication. After lysis, the cells were centrifuged at 40,000 rpm for 40 minutes at 4°C. The supernatant was collected to obtain the cell lysate. The cell lysate was purified using equilibrated glutathione affinity resin (Genescript) and washed three times. The GST-tagged ATF6 N protein was eluted with elution buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mg / ml reduced glutathione). Reduced glutathione was removed from the eluted protein using a Sephadex G-25 desalting column (Cytiva) to obtain the GST-ATF6 N fusion protein.

[0163] Example 5 Association between ATF6 N protein and PMP70

[0164] Experimental methods:

[0165] The ATF6 N protein gene was amplified from HeLa cDNA and cloned into the pEGFP-C1 plasmid vector by Gibson assembly and placed between the BamHI and EcoRI sites. The pEGFP-C1 plasmid vector was transfected into HeLa cells for 24 h. HeLa cells were treated with Ceapin-A7 or DMSO, and then fixed and stained with PMP70 (Alexa Fluor 555) for immunofluorescence analysis.

[0166] The ATF6 N protein gene was amplified from HeLa cDNA and cloned into the pEGFP-C1 plasmid vector by Gibson assembly between the BamHI and EcoRI sites. HEK293T cells were transfected with the pEGFP-C1 plasmid vector for 24 hours and then treated with 6 μM DMSO or Ceapin-A7 for 24 hours. HEK293T cells were lysed, and the cell lysates were immunoprecipitated with a GFP antibody and immunoblotted with the indicated antibodies.

[0167] Experimental results:

[0168] As shown in Figure 1A, immunofluorescence analysis showed that GFP-ATF6 N protein was expressed in the cytoplasm in DMSO-treated cells and colocalized with PMP70 in Ceapin-A7-treated cells. GFP-ATF6 N protein was then immunoprecipitated from cell lysates containing detergent. As shown in Figure 1B, interaction between GFP-ATF6 N protein and PMP70 was observed regardless of the presence or absence of Ceapin-A7.

[0169] Example 6 GST-ATF6 N fusion protein binds to PMP70 and ABCD1

[0170] Experimental methods:

[0171] The GST-ATF6 N fusion protein was obtained by expression and purification of the GST-ATF6 N fusion protein in Example 4.

[0172] 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 using HeLa lysis buffer (20 mM Tris, 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. GST-ATF6 N fusion protein was added to the supernatant to obtain a protein mixture of PMP70 and the fusion protein, and a protein mixture of ABCD1 and the fusion protein. Both protein mixtures were incubated with glutathione affinity resin for 2 hours at 4°C. The glutathione affinity resin was then collected, washed three times with wash buffer, and subjected to immunoblotting.

[0173] In vitro pull-down assay: Flag-PMP70 and Flag-ABCD1 obtained in Example 3 were mixed with GST-ATF6N fusion protein to obtain two protein mixtures of Flag-PMP70 and fusion protein and one protein mixture of Flag-ABCD1 and fusion protein, and incubated at 4°C for 30 minutes in a buffer containing 25mM pH 7.5 Tris-HCl and 150mM NaCl. Glutathione affinity resin or anti-flag M2 affinity gel was added to the two protein mixtures and incubated at 4°C for 1 hour. The glutathione affinity resin or anti-flag M2 affinity gel was then collected, washed three times with wash buffer (25mM pH 7.5 Tris-HCl and 150mM NaCl), and subjected to SDS-PAGE or immunoblotting.

[0174] Immunoprecipitation and immunoblotting: Protein mixtures containing Flag-PMP70 and fusion proteins, as well as Flag-ABCD1 and fusion proteins, were immunoprecipitated with GFP antibodies and protein G beads. The mixtures were incubated with rotation overnight at 4°C. The immune complexes were pelleted by centrifugation and washed three times with lysis buffer. The immune complexes were boiled with SDS loading buffer and heated at 95°C for 10 minutes. The supernatant was subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, IPVH00010). The membranes were probed with the primary antibody and the indicated secondary antibody conjugated to horseradish peroxidase. Signals were detected using enhanced chemiluminescence substrate (Epizyme) according to the manufacturer's instructions.

[0175] HeLa cell lysate was incubated with increasing gradients of GST-ATF6 N protein, and then treated with 6 μM DMSO or Ceapin-A7 for 2 h, and the above pull-down assay, co-immunoprecipitation, and immunoblotting experiments were repeated.

[0176] Experimental results:

[0177] In vivo pull-down assay: As shown in Figures 1D-E, the GST-ATF6 N protein obtained in Example 4 pulled down PMP70 from HeLa cell lysates, demonstrating an interaction between GST-ATF6 N protein and PMP70. This interaction was enhanced when more GST-ATF6 N protein was added to the cell lysate. Furthermore, in HeLa cell lysates containing high levels of GST-ATF6 N protein, the peroxisomal membrane protein PEX13 was detected in the GST-ATF6 N protein pull-down complex (Figure 1E). As shown in Figure 1F, immunoblot analysis revealed that GST-ATF6 N protein interacted with both PMP70 and ABCD1.

[0178] In vitro pull-down assays: As shown in Figures 1G-I, purified FLAG-ABCD1 and FLAG-PMP70 proteins were able to bind to GST-ATF6 N protein in vitro. As shown in Figures 1J-K, GST pull-down assays further demonstrated that GST-ATF6N protein did not bind to other peroxisomal proteins, such as PEX19 and PEX5.

[0179] The above results indicate that ATF6 N protein can specifically interact with ABCD1 and PMP70 regardless of the presence of Ceapin-A7.

[0180] Example 7 GST-ATF6 N protein binding affinity chromatography separation of peroxisomes

[0181] Experimental methods:

[0182] For HeLa cells, wash the cells three times with PBS, resuspend in PBS, and disrupt them using a 7ml Dounce homogenizer with Pestle B, using 80 strokes. After homogenization, centrifuge at 800 x g for 10 minutes at 4°C. Transfer the supernatant to a fresh centrifuge tube and centrifuge at 8000 x g for 10 minutes at 4°C. Transfer the supernatant to a fresh centrifuge tube to obtain the crude peroxisome sample.

[0183] For mouse tissue, the specific tissue was cut into small pieces and washed three times with PBS. The tissue pieces were suspended in PBS and broken up using a 7 ml Dounce homogenizer with Pestle B, followed by the same centrifugation steps as for HeLa cells.

[0184] GST-ATF6 N fusion protein was obtained according to the expression and purification of GST-ATF6 N fusion protein in Example 4.

[0185] Resuspend the glutathione affinity resin and transfer 400 μl of resin (50% suspension) to a 3 ml gravity column; equilibrate the precipitated resin with 10 ml of PBS, add 4 mg of 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 three times with PBS; add the crude peroxisomal 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 and wash three 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 four more times and combine the five elution fractions; the isolated and purified peroxisomes can be concentrated using a 100 kDa concentrator tube (Millipore).

[0186] Experimental results:

[0187] Peroxisomes were isolated and purified by the above method, and identification showed that 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 separate and purify peroxisomes from other cellular components.

[0188] Example 8 Purity and efficiency of peroxisomes isolated and purified by the present invention

[0189] Experimental methods:

[0190] A HeLa cell line (CSS cell line) stably expressing BFP-SKL, COXIV-EGFP and mCherry-SEC61B was constructed, and the peroxisome matrix, mitochondrial membrane and endoplasmic reticulum membrane were fluorescently labeled respectively. The specific operation was as follows: HEK293T cells were cultured, and HEK293T cells were transfected with lentiviral vectors pCMV-dR8.91 and pMD2G respectively. The cell culture medium containing lentivirus was harvested 48 hours after transfection. The viral supernatant was filtered with a 0.45μm filter membrane. BFP-SKL, COX-EGFP, SEC61b-mCherry lentivirus were infected with HeLa cells at a confluence of 50%. BFP was isolated by flow cytometry fluorescence sorting technology. + , GFP + 、mCherry +Triple-positive cells were sorted into 96-well plates, with one cell per well selected for monoclonal selection. Confocal microscopy was used to examine the expression and localization of BFP, GFP, and mCherry. Peroxisomes were isolated and purified using the method of Example 7. During the isolation and purification process of Example 7, the ratio of the three fluorescent signals in the peroxisomes isolated and purified in Example 7 was monitored by immunofluorescence. The abundance of peroxisomal-associated marker proteins was analyzed by immunoblotting.

[0191] Experimental results:

[0192] As shown in Figures 2B-C, the proportion of BFP-SKL gradually increased, while the proportions of COXIV-EGFP and mCherry-SEC61B gradually decreased. In the peroxisomes isolated and purified in Example 7, only BFP-SKL fluorescence was observed, while COXIV-EGFP and mCherry-SEC61B fluorescence was almost absent. This indicates that the content of the peroxisomal matrix isolated and purified in Example 7 gradually increased, and the purified peroxisomes were of high purity, containing almost no other impurities. As shown in Figure 2D, the presence of various organelles was detected in the lysate of WT cells, while PMP70 and ABCD1 were not detected in the lysate of PEX19 KO cells. Peroxisomal membrane proteins and matrix proteins were detected in the peroxisomal samples of WT cells, but not in the peroxisomal samples of PEX19 KO cells. In both WT and PEX19 KO cells, the peroxisomal samples isolated and purified in Example 7 contained almost no other organelle markers.

[0193] Example 9 Evaluation of the activity of peroxisomes isolated and purified by the method of the present invention

[0194] Experimental methods:

[0195] Example 7 The isolated and purified peroxisomes were lysed in 200 μl of extract containing 1% Triton X-100 to obtain a lysate. The lysate was centrifuged at 13,000 x g and 4° C. for 10 minutes, and the supernatant was transferred to a new tube. Catalase activity: The supernatant was detected using the CheKine Catalase (CAT) Activity Assay Kit (Abbkine). Glutamate dehydrogenase (GDH) activity: The supernatant was detected using the GDH Activity Assay Kit (Sangon Biotech). Carboxylesterase (CarE) activity: The supernatant was detected using the Carboxylesterase (CarE) Activity Assay Kit (Sangon Biotech). Calculation was performed according to the manufacturer's method. The enzyme activity was normalized to the protein concentration in the supernatant using the BCA method.

[0196] Experimental results:

[0197] The enzymatic activities of peroxisomal, mitochondrial, and endoplasmic reticulum marker enzymes were measured in the peroxisomes isolated and purified in Example 7. As shown in Figures 2E-G, catalase activity was detected only in the peroxisomes of WT cells.

[0198] Example 10 Comparison of the method for isolating and purifying catalase bodies of the present invention with conventional density gradient centrifugation

[0199] Experimental methods:

[0200] For traditional density gradient centrifugation to isolate peroxisomes, use the Peroxisome Isolation Kit (PEROX1, Sigma) according to the manufacturer's instructions: Harvest HeLa cells and resuspend them in Peroxisome Extraction Buffer. Transfer the cell suspension to a Dounce homogenizer and homogenize with Pestle B for 80 strokes. Centrifuge the homogenized cell suspension at 1000 x g for 10 minutes at 4°C. Transfer the supernatant to a fresh centrifuge tube and centrifuge at 2000 x g for 10 minutes at 4°C. Centrifuge the supernatant at 25,000 x g for 20 minutes at 4°C. Discard the supernatant and resuspend the pellet in Peroxisome Extraction Buffer.

[0201] For the density gradient centrifugation step, use a gradient mixer immediately before use and pour a linear Nykodenz density gradient ranging from 1.14 to 1.20 g / ml. Layer the pellet suspension on top of the Nykodenz gradient and seal the tube. Centrifuge at 100,000 x g for 3 hours at 4°C. Fractions are collected using a Biocomp system.

[0202] Experimental results: The methods of Example 7 and this example were used to isolate peroxisomes from the CSS cell line, respectively. As shown in Figure 3A, the peroxisomes separated 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. In the peroxisomes separated and purified in this example, three fluorescent signals were detected in the part with the most BFP-SKL signals. It was found that a certain number of SEC61B-mCherry signals were present in this part, and both SEC61B-mCherry and BFP-SKL signals were clustered. As shown in Figure 3B, immunoblotting showed that the peroxisomes separated and purified by density gradient centrifugation in this example were also contaminated by other organelles. However, the peroxisomes separated and purified in Example 7 rarely contained components of other organelles.

[0203] Example 11 Flow cytometric analysis of peroxisomes

[0204] Experimental Methods: Peroxisomes isolated and purified from WT cells and cells stably expressing EGFP-SKL using the method of Example 7 were compared. Specifically, peroxisomes isolated from WT cells or GFP-SKL cells using Example 7 were suspended in PBS. The WT HeLa cells and GFP-SKL cells were fixed by incubation with primary and secondary antibodies. The peroxisomes isolated and purified from WT HeLa cells and GFP-SKL cells were stained with the PEX14 antibody (Alexa Fluor 555). After staining, the peroxisomes were eluted using CytoFLEX (Beckman Coulter) and analyzed. Data were analyzed using FlowJo version 10 software.

[0205] Experimental Results: As shown in Figure 3C, flow cytometry analysis showed that the EGFP signal of EGFP-SKL peroxisomes isolated and purified using the method of Example 7 was much higher than that of WT peroxisomes. Figures 3D and E show peroxisomes stained with PEX14 antibodies in flow cytometry.

[0206] Example 12 Transmission electron microscope observation

[0207] Experimental Method: The peroxisomes isolated and purified in Example 7 were observed using a transmission electron microscope. Specifically, 4 μL of the peroxisomal solution isolated and purified in Example 7 was applied to a Quantifoil R2 / 2 200-mesh porous carbon gold grid (Quantifoil, Micro Tools GmbH, Germany) and illuminated for 45 seconds using a Solarus 950 (Gatan, USA) in a H2 / O2 atmosphere. The grid was immediately blotted for 5.0 seconds from the back at a horizontal blot position of 44 mm and a vertical blot position of 2.5 mm at 4°C and 90% humidity. The grid was then immersed in liquid ethylmethane using a Leica EM GP2 automated blot freezer (Leica Microsystems GmbH, Austria).

[0208] The grids were 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 at a nominal magnification of 29,000× (pixel size ), the defocus is 2.5μm, and the total dose is

[0209] Experimental results: As shown in Figure 3F, the cryo-transmission electron microscopy (cryo-TEM) analysis results showed the high resolution of the peroxisomes isolated and purified in Example 7, showing the integrity of the peroxisomal 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.

[0210] Example 13 Analysis of the peroxisomal proteome of mammalian WT HeLa cells

[0211] Experimental Methods: A peroxisomal protein reference set of 103 peroxisomal proteins was obtained from literature / databases (UniProt). The peroxisomal protein reference set was identified by mass spectrometry. Peroxisomes isolated and purified from mammalian WT HeLa cells and PEX19 KO cells as described in Example 7 were analyzed using label-free quantitative mass spectrometry. Peroxisomes purified from PEX19 KO cells were used as a control pellet.

[0212] Experimental Results: Peroxisomes isolated and purified from mammalian WT HeLa cells and PEX19 KO cells according to Example 7 are shown in Figure 4A. As shown in Figures 4B and 4D, a total of 280 peroxisomal proteins were identified from mammalian WT HeLa cells, of which 72 belonged to the aforementioned peroxisomal protein reference set and 208 were novel peroxisomal candidate proteins. As shown in Figure 4C, gene ontology (GO) enrichment analysis and KEGG pathway analysis revealed that the 280 peroxisomal proteins possess peroxisome-related functions and peroxisomal isoform-related functions, such as fatty acid metabolism and lipid oxidation. As shown in Figure 4E, a total of 208 peroxisomal proteins were novel peroxisomal candidate proteins, enriched in fatty acid metabolism and glycosylation processes, and were associated with fatty acid metabolism and glycosylation. As shown in Figure 4F, among the 208 novel peroxisomal candidate proteins, CTP1A and AKAP1 were localized to mitochondria, and CYB5R1 was localized to the endoplasmic reticulum. When YFP-tagged candidates were transfected into HeLa cells, CYB5R1 signals were observed to co-localize with mitochondria and peroxisomes. This example illustrates that the peroxisomes isolated and purified in Example 7 can be used for subsequent protein profiling analysis.

[0213] Example 14 Tissue-specific analysis of mouse peroxisomal proteome

[0214] Experimental Methods: Peroxisomes were isolated and purified from mouse brain, liver, and kidney using the method described in Example 7. To investigate the heterogeneity of peroxisomal proteins in different mouse tissues, a label-free proteomic analysis was developed to analyze and compare the peroxisomal proteomes isolated and purified from mouse brain, liver, and kidney (n=3).

[0215] As shown in Figure 5A-B, in mass spectrometry analysis, 3907 oxisome proteins were identified in brain peroxisomes, accounting for 70.9% of the reference peroxisomal proteins; 3649 oxisome proteins were identified in liver peroxisomes, accounting for 89.3% of the reference peroxisomal proteins; and 5321 oxisome proteins were identified in kidney peroxisomes, accounting for 94.2% of the reference peroxisomal proteins.

[0216] As shown in Figure 5A, 2333 peroxisomal proteins were shared by the three tissues. As shown in Figure 5C, the expression of most peroxisomal proteins did not differ significantly among the three tissues. As shown in Figures 5C and 5G, some peroxisomal proteins exhibited tissue specificity, such as Syt7, which was found only in the brain, Mpv17l, which was found only in the kidney, and Hao1 and Urad, which were found only in the liver. Some proteins were not detected in some tissues, such as Tmem35a and Dao, which 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. As shown in Figure 5D, KEGG pathway and GO Term Process analysis confirmed the enrichment of peroxisome-related processes. As shown in Figures 5C and 5G, some proteins were not detected in some tissues, including Tmem35a and Dao, which 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, indicating that the peroxisomes separated and purified in Example 7 can be used for subsequent protein profiling analysis.

[0217] Example 15 Data Statistics

[0218] Quantitative data were expressed as mean ± standard deviation, and the P value was calculated using unpaired two-tailed t test in GraphPad Prism to evaluate the significant difference between the two groups.

[0219] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may 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 separating and purifying 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 separating 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 the 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 one 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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