Purification method of GSL5 protein with disease-resistant and disease-susceptible dual functions
By fusing an expression vector containing Strep II and His tags to the end of the GSL5 gene, and combining resuspension, disruption, centrifugation, and two-step affinity chromatography, we achieved highly efficient purification of the GSL5 protein, solving the problem of its high purification difficulty and significantly improving purification efficiency.
Patent Information
- Application Number
- CN202511012563.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-22
- Publication Date
- 2025-12-09
AI Technical Summary
The purification methods for GSL5 protein in existing technologies are difficult, which limits the research on its mechanism and the exploration of its application potential as a dual-function gene for disease resistance and susceptibility.
An expression vector with Strep II and His tags fused to the ends of the GSL5 gene was used. After expression in eukaryotic cells, high-purity GSL5 protein samples were obtained by resuspension, disruption, centrifugation, affinity chromatography using Strep packing material and nickel column packing material.
This method achieves highly efficient purification of GSL5 protein, significantly improving preparation and purification efficiency. It optimizes the conditions and parameters in the purification process, and in particular, avoids the cumbersome steps of conventional protein purification processes through a two-step affinity chromatography purification method.
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Figure CN121087008A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of protein purification technology, and in particular relates to a purification method for GSL5 protein with dual functions of disease resistance and disease susceptibility. Background Technology
[0002] GSL5, a disease resistance gene, encodes a callosine synthase that plays a crucial role in plant cell wall construction, pollen maturation, and other growth and development processes. It also plays a role in the dynamic reinforcement of the cell wall during plant resistance to pathogen infection. In Arabidopsis lines overexpressing GSL5, a significantly thickened callosine layer is present at the pathogen infection site, completely inhibiting the penetration and spread of powdery mildew.
[0003] GSL5, as a susceptibility gene, encodes a product that mediates infection of the host by various pathogens, including powdery mildew, clubroot fungi, and Phytophthora (the pathogen of late blight). Previous studies have shown that in Arabidopsis mutants with the GSL5 gene knocked out, the early penetration rate of pathogens is significantly increased, but unexpectedly, the later spread of powdery mildew is significantly suppressed, thus the GSL5 mutants ultimately exhibit a resistant phenotype. This "susceptibility followed by resistance" phenotype is species-conserved; GSL5-mutant tomato plants show significantly higher resistance to powdery mildew and Phytophthora than the wild type, and knocking down the GSL5 gene in potatoes confers late blight tolerance. Our latest research found that GSL5 gene knockout in Arabidopsis, rapeseed, Chinese cabbage, and olive all resulted in a broad-spectrum, high-resistance phenotype against clubroot fungi.
[0004] The GSL5 gene was first cloned in the 1990s. Encoding 1780 amino acids, it is predicted to contain 15 transmembrane domains. Its complex spatial structure makes purification extremely difficult, and no purification methods have been reported in the past 30 years, significantly limiting research into its mechanism of action and potential applications as a dual-function gene for disease resistance and susceptibility. Therefore, developing efficient, simple, and suitable purification methods for GSL5 is crucial for a deeper understanding of its mechanism of action and for exploring its application potential. Summary of the Invention
[0005] In view of this, the present invention discloses a purification method for GSL5 protein with dual functions of disease resistance and disease susceptibility.
[0006] The present invention adopts the following technical solution:
[0007] A method for purifying GSL5 protein with dual disease resistance and disease susceptibility functions, the purification method comprising the following steps:
[0008] S1. Construct an expression vector with two affinity tags fused to the end of the GSL5 gene, the affinity tags including a StrepII tag and a His tag, and express the expression vector in eukaryotic cells;
[0009] S2. Collect cells expressing the GSL5 protein. The cells are subjected to resuspension, high-pressure disruption, detergent extraction and high-speed centrifugation in sequence to obtain protein solution.
[0010] S3. The protein solution is purified by affinity chromatography using Strep packing material and eluted with biotin to obtain affinity eluent 1.
[0011] S4. The eluent is purified by affinity chromatography using a nickel column packing material to obtain high-purity GSL5 protein.
[0012] Further, step S1 includes: obtaining a fragment of the GSL5 gene fused with a Strep II tag at the end by means of PCR amplification technology, and ligating the fragment into a eukaryotic expression vector containing a C-terminal His tag.
[0013] Furthermore, the His tag is an extended version containing 10 histidine residues; the amino acid sequence of the Strep II tag is WSHPQFEKGGGSGGGSGGSAWSHPQFEK.
[0014] Further, the eukaryotic cells in step S1 are mammalian Expi293 cells or insect SF9 cells; the culture temperature of the Expi293 cells is 37°C, the carbon dioxide concentration is 5%, and the shaking speed is 100 rpm; the culture temperature of the SF9 cells is 27°C, and the shaking speed is 100 rpm.
[0015] Further, the resuspension buffer used in step S2 comprises: 100 mM Tris-HCl buffer, 300 mM sodium chloride, and 20% glycerol; the detergent in step S2 is n-Dodecyl-β-D-maltopyranoside (DDM) or lauryl maltoseneopentyl glycerol (LMNG), and the concentration of the detergent is 1%-2%.
[0016] Furthermore, the Strep filler mentioned in step S3 is Strep-Tactin XT 4Flow.
[0017] Furthermore, the specific steps of affinity chromatography purification described in step S3 include equilibration, incubation, washing, and elution.
[0018] Furthermore, the pH of the equilibration buffer and the washing buffer used in the affinity chromatography purification process described in step S3 is 7.4-8.0; biotin with a final concentration of 25 mM is added to the equilibration buffer to form the elution buffer.
[0019] Furthermore, the nickel column packing material in step S4 is NI-NTA Agarose, and the specific steps of affinity chromatography purification in step S4 include equilibration, sample loading, washing, and elution.
[0020] Furthermore, the pH value of the equilibration buffer used in the affinity chromatography purification process in step S4 is 7.4-8.0. Imidazole with a final concentration of 15-30 mM is added to the equilibration buffer to form an elution buffer, and imidazole with a final concentration of 250-500 mM is added to the equilibration buffer to form an elution buffer.
[0021] The beneficial effects of this invention are:
[0022] The present invention provides a method for purifying GSL5 protein with dual functions of disease resistance and disease susceptibility, comprising the following steps: collecting cells containing GSL5 protein, resuspending, disrupting, extracting, and centrifuging to obtain a protein solution; performing a primary affinity chromatography on the protein solution using StrepII packing material to obtain affinity eluent one; and performing a secondary affinity chromatography on the affinity eluent one using a nickel column packing material to obtain a purified GSL5 protein sample.
[0023] This invention constructs an expression vector fused with two affinity tags at the end of the GSL5 gene, namely the Strep II tag and the His tag, and then expresses the expression vector in eukaryotic cells; and achieves efficient purification of GSL5 protein by performing two affinity chromatography steps on the protein solution, significantly improving the efficiency of GSL5 protein preparation and purification.
[0024] This invention systematically describes the steps for purifying GSL5 protein for the first time and significantly optimizes the conditions and parameters in the purification process. In particular, it systematically tested the composition of the resuspension buffer. When the sodium chloride concentration is 300 mM and the glycerol concentration is 20%, the target protein (GSL5 protein) sample changes from an insoluble precipitate state to a soluble state, which can significantly improve the resuspension effect and increase the purification efficiency. At the same time, the two-step affinity chromatography purification method avoids the conventional protein purification process of "crude purity-intermediate purity-refined purity", which significantly improves the purification efficiency. Attached Figure Description
[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0026] Figure 1: A schematic diagram of the GSL5 protein purification method of the present invention;
[0027] Figure 2 : Schematic diagram of SDS-PAGE electrophoresis of GSL5 protein after two-step affinity purification in this invention;
[0028] Figure 3 : Schematic diagram of SDS-PAGE electrophoresis of GSL5 protein purified by Strep II affinity without optimization of resuspension buffer in this invention. Detailed Implementation
[0029] To better understand the technical solution of the present invention, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
[0030] It should be understood that the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
[0031] Example 1
[0032] A purification method for GSL5 protein with dual disease resistance and disease susceptibility functions, such as... Figure 1 As shown, the purification method includes the following steps:
[0033] S1. Construct an expression vector with two affinity tags fused to the end of the GSL5 gene, the affinity tags including a StrepII tag and a His tag, and express the expression vector in eukaryotic cells;
[0034] S2. Collect cells expressing the GSL5 protein. The cells are subjected to resuspension, high-pressure disruption, detergent extraction and high-speed centrifugation in sequence to obtain protein solution.
[0035] S3. The protein solution is purified by affinity chromatography using Strep packing material and eluted with biotin to obtain affinity eluent 1.
[0036] S4. The eluent is purified by affinity chromatography using a nickel column to obtain high-purity GSL5 protein. Figure 2 As shown, Figure 2 This is an SDS-PAGE electrophoresis image of GSL5 after two-step affinity purification.
[0037] Further, step S1 includes: obtaining a fragment of the GSL5 gene fused with a Strep II tag at the end by means of PCR amplification technology, and ligating the fragment into a eukaryotic expression vector containing a C-terminal His tag.
[0038] Furthermore, the His tag is an extended version containing 10 histidine residues; the amino acid sequence of the Strep II tag is WSHPQFEKGGGSGGGSGGSAWSHPQFEK.
[0039] Further, the eukaryotic cells mentioned in step S1 are mammalian Expi293 cells or insect SF9 cells; the culture temperature of Expi293 cells is 37°C, the carbon dioxide concentration is 5%, and the shaking speed is 100 rpm; the culture temperature of SF9 cells is 27°C, and the shaking speed is 100 rpm.
[0040] Further, the resuspension buffer used in step S2 comprises: 100 mM Tris-HCl buffer, 300 mM sodium chloride, and 20% glycerol; the detergent in step S2 is n-Dodecyl-β-D-maltopyranoside (DDM) or lauryl maltose-neopentyl glycerol (LMNG), with a concentration of 1%-2%; the cell collection time in step S2 is 60 hours post-transfection; the pH of the resuspension buffer used in step S2 is 7.8. Figure 3 As shown, Figure 3 When resuspended with a different resuspension buffer (unoptimized resuspension buffer), the SDS-PAGE electrophoresis image of GSL5 after the first step of affinity purification shows that the target protein GSL5 is always in the precipitate (lane 1), and there are only impurities and no target protein in the elution buffer (lane 5).
[0041] Furthermore, the Strep packing material mentioned in step S3 is Strep-Tactin XT 4Flow. Strep-Tactin XT 4Flow is a protein purification packing material.
[0042] Furthermore, the specific steps of affinity chromatography purification described in step S3 include equilibration, incubation, washing, and elution.
[0043] Further, the pH of the equilibration buffer and washing buffer used in the affinity chromatography purification process in step S3 is 7.8. The equilibration buffer and washing buffer include 50 mM Tris-HCl buffer, 150 mM sodium chloride, and 0.02% n-dodecyl-β-D-maltopyranoside (DDM) or lauryl maltose neopentyl glycol (LMNG). An elution buffer is prepared by adding biotin to the equilibration buffer to a final concentration of 25 mM. The incubation temperature in step S3 is 4°C, and the incubation time is 1-2 hours. The equilibration buffer and washing buffer used in the affinity chromatography purification process in step S3 are the same.
[0044] Furthermore, the nickel column packing material in step S4 is NI-NTA Agarose, and the specific steps of affinity chromatography purification in step S4 include equilibration, sample loading, washing, and elution.
[0045] Further, the equilibration buffer used in the affinity chromatography purification process in step S4 has a pH of 7.8. The equilibration buffer includes a 25 mM Tris-HCl buffer, a 150 mM sodium chloride, and a 0.02% concentration of n-Dodecyl-β-D-maltopyranoside (DDM) or lauryl maltose neopentyl glycol (LMNG). An imidazole with a final concentration of 25 mM is added to the equilibration buffer to form an elution buffer, and an imidazole with a final concentration of 300 mM is added to the equilibration buffer to form an elution buffer.
[0046] Example 2
[0047] The purification method for GSL5 protein in this embodiment includes the following steps:
[0048] (1) Collect SF9 insect cells expressing GSL5 protein, and obtain protein solution after resuspension, high pressure disruption, detergent extraction and centrifugation;
[0049] The resuspension buffer has a pH of 8.0 and comprises: 100 mM Tris-HCl buffer, 300 mM sodium chloride, and 20% glycerol. The high-pressure crusher operates at 1000 bar and 4°C. The detergent used in the extraction process is n-Dodecyl-β-D-maltopyranoside (DDM) with a final concentration of 2%. The centrifugation conditions are 14000 rpm, 4°C, and 40 minutes.
[0050] (2) The protein solution obtained in step (1) is subjected to affinity chromatography using Strep II packing material to obtain affinity eluent 1; Strep II packing material is Strep-Tactin XT 4Flow protein purification packing material.
[0051] The StrepII affinity chromatography includes equilibration, incubation, washing, and elution. The equilibration and washing buffers are at pH 8.0 and contain 50 mM Tris-HCl buffer, 150 mM sodium chloride, and 0.02% n-Dodecyl-β-D-maltopyranoside (DDM). The elution buffer is at pH 7.5 and contains 50 mM Tris-HCl buffer, 150 mM sodium chloride, 0.02% n-Dodecyl-β-D-maltopyranoside (DDM), and 25 mM biotin.
[0052] (3) The affinity eluent obtained in step (2) is subjected to affinity chromatography again using a nickel column packing to obtain the purified GSL5 protein sample.
[0053] The nickel column affinity chromatography includes equilibration, sample loading, washing, and elution. The equilibration buffer has a pH of 8.0 and includes 25 mM Tris-HCl buffer, 150 mM sodium chloride, and 0.02% n-Dodecyl-β-D-maltopyranoside (DDM). The washing buffer has a pH of 8.0 and includes 25 mM Tris-HCl buffer, 150 mM sodium chloride, 0.02% n-Dodecyl-β-D-maltopyranoside (DDM), and 15 mM imidazole. The elution buffer has a pH of 8.0 and includes 25 mM Tris-HCl buffer, 150 mM sodium chloride, 0.02% n-Dodecyl-β-D-maltopyranoside (DDM), and 250 mM imidazole.
[0054] Example 3
[0055] The purification method for GSL5 protein in this embodiment includes the following steps:
[0056] (1) Collect Expi293 mammalian cells expressing GSL5 protein, and obtain protein solution after resuspension, high pressure disruption, detergent extraction and centrifugation;
[0057] The resuspension buffer has a pH of 7.4 and includes 100 mM Tris-HCl buffer, 300 mM sodium chloride, and 20% glycerol. The high-pressure crusher operates at 1000 bar and 4°C. The detergent used in the extraction process is n-Dodecyl-β-D-maltopyranoside (DDM) with a final concentration of 1%. The centrifugation conditions are 14000 rpm, 4°C, and 40 minutes.
[0058] (2) Perform affinity chromatography on the protein solution obtained in step (1) using Strep II packing material to obtain affinity eluent 1;
[0059] The StrepII affinity chromatography includes equilibration, incubation, washing, and elution. The equilibration and washing buffers are at pH 7.4 and contain 50 mM Tris-HCl buffer, 150 mM sodium chloride, and 0.02% n-Dodecyl-β-D-maltopyranoside (DDM). The elution buffer is at pH 7.5 and contains 50 mM Tris-HCl buffer, 150 mM sodium chloride, 0.02% n-Dodecyl-β-D-maltopyranoside (DDM), and 25 mM biotin.
[0060] (3) The affinity eluent obtained in step (2) is subjected to affinity chromatography again using a nickel column packing to obtain the purified GSL5 protein sample.
[0061] The nickel column affinity chromatography includes equilibration, sample loading, washing, and elution. The equilibration buffer has a pH of 7.4 and comprises 25 mM Tris-HCl buffer, 150 mM sodium chloride, and 0.02% n-Dodecyl-β-D-maltopyranoside (DDM). The washing buffer has a pH of 7.4 and comprises 25 mM Tris-HCl buffer, 150 mM sodium chloride, 0.02% n-Dodecyl-β-D-maltopyranoside (DDM), and 20 mM imidazole. The elution buffer has a pH of 7.4 and includes 25 mM Tris-HCl buffer, 150 mM sodium chloride, 0.02% n-Dodecyl-β-D-maltopyranoside (DDM), and 350 mM imidazole.
[0062] Example 4
[0063] The purification method for GSL5 protein in this embodiment includes the following steps:
[0064] (1) Collect SF9 insect cells expressing GSL5 protein, and obtain protein solution after resuspension, high pressure disruption, extraction and centrifugation;
[0065] The resuspension buffer has a pH of 7.8 and includes 100 mM Tris-HCl buffer, 300 mM sodium chloride, and 20% glycerol. The high-pressure grinder operates at 1000 bar and 4°C. The detergent used in the extraction process is 2% lauryl maltose neopentyl glycol (LMNG). The centrifugation conditions are 14000 rpm, 4°C, and 40 minutes.
[0066] (2) Perform affinity chromatography on the protein solution obtained in step (1) using Strep II packing material to obtain affinity eluent 1;
[0067] The StrepII affinity chromatography includes equilibration, incubation, washing, and elution. The equilibration and washing buffers have a pH of 7.8 and contain 50 mM Tris-HCl buffer, 150 mM sodium chloride, and 0.02% lauryl maltose neopentyl glycol (LMNG). The elution buffer has a pH of 7.5 and contains 50 mM Tris-HCl buffer, 150 mM sodium chloride, 0.02% lauryl maltose neopentyl glycol (LMNG), and 25 mM biotin.
[0068] (3) The affinity eluent obtained in step (2) is subjected to affinity chromatography again using a nickel column packing to obtain the purified GSL5 protein sample.
[0069] The nickel column affinity chromatography includes equilibration, sample loading, washing, and elution. The equilibration buffer has a pH of 7.8 and comprises 25 mM Tris-HCl buffer, 150 mM sodium chloride, and 0.02% lauryl maltose neopentyl glycol (LMNG). The washing buffer has a pH of 7.8 and comprises 25 mM Tris-HCl buffer, 150 mM sodium chloride, 0.02% lauryl maltose neopentyl glycol (LMNG), and 30 mM imidazole. The elution buffer has a pH of 7.8 and includes 25 mM Tris-HCl buffer, 150 mM sodium chloride, 0.02% lauryl maltose neopentyl glycol (LMNG), and 500 mM imidazole.
[0070] Example 5
[0071] A purification method for GSL5 protein with dual disease resistance and disease susceptibility functions, such as... Figure 1 As shown, it includes the following steps:
[0072] (1) Collect cell bodies containing GSL5, and obtain protein solution after resuspension, high pressure disruption, extraction and centrifugation;
[0073] (2) Perform affinity chromatography on the protein solution obtained in step (1) using Strep II packing material to obtain affinity eluent 1;
[0074] (3) The affinity eluent obtained in step (2) is subjected to affinity chromatography again using a nickel column packing to obtain the purified GSL5 protein sample.
[0075] Preferably, in step (1), the resuspension buffer used has a pH of 7.8 and includes a 100 mM Tris-HCl buffer, a 300 mM sodium chloride, and a 20% glycerol.
[0076] Preferably, in step (1), the pressure of the high-pressure crusher used is 1000 bar and the temperature is 4°C.
[0077] Preferably, in step (1), the reagent used in the extraction process is the detergent n-Dodecyl-β-D-maltopyranoside (DDM) or lauryl maltoseneopentyl glycol (LMNG), with a final concentration of 1.5%.
[0078] Preferably, in step (1), the centrifugation conditions are: a rotation speed of 14,000 rpm, a temperature of 4°C, and a centrifugation time of 40 minutes to 1 hour.
[0079] Preferably, in step (2), the affinity chromatography process includes equilibration, incubation, washing, and elution; the equilibration buffer and washing buffer used in step (2) have a pH of 7.8 and include a 40 mM Tris-HCl buffer, a 150 mM sodium chloride, and a 0.02% concentration of n-Dodecyl-β-D-maltopyranoside (DDM) or lauryl maltose neopentyl glycol (LMNG); the elution buffer is the equilibration buffer with the addition of biotin to a final concentration of 25-50 mM.
[0080] Preferably, in step (3), the nickel column affinity chromatography process includes equilibration, sample loading, washing, and elution; the equilibration buffer used in step (3) has a pH of 7.8 and includes 40 mM Tris-HCl buffer, 150 mM sodium chloride, and 0.02% n-Dodecyl-β-D-maltopyranoside (DDM) or lauryl maltose neopentyl glycol (LMNG); the washing buffer used in step (3) has a pH of 7.8 and includes 40 mM Tris-HCl buffer, 150 mM sodium chloride, and 0.02% n-Dodecyl-β-D-maltopyranoside (DDM) or lauryl maltose neopentyl glycol (LMNG). The elution buffer used in step (3) has a pH of 7.8 and includes 40 mM Tris-HCl buffer, 150 mM sodium chloride, 0.02% n-Dodecyl-β-D-maltopyranoside (DDM) or lauryl maltose neopentyl glycol (LMNG), and 350 mM imidazole.
[0081] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.
Claims
1. A method for purifying a GSL5 protein having a dual function of disease resistance and susceptibility, characterized by, The purification method comprises the following steps: S1, constructing an expression vector with two affinity tags fused at the end of the GSL5 gene, the affinity tags comprising a Strep II tag and a His tag, and expressing the expression vector in eukaryotic cells; S2, collecting cells expressing the GSL5 protein, and obtaining a protein solution after the cells sequentially undergo resuspension, high-pressure crushing, detergent extraction and high-speed centrifugation; S3, subjecting the protein solution to affinity chromatography purification with Strep packing material, and eluting with biotin to obtain an affinity eluate I; S4, subjecting the eluate I to affinity chromatography purification with nickel column packing material to obtain the GSL5 protein with high purity.
2. The purification method of claim 1, wherein, Step S1 comprises: obtaining a fragment with a Strep II tag fused at the end of the GSL5 gene by PCR amplification technology, and connecting the fragment to a eukaryotic expression vector containing a C-terminal His tag.
3. The purification method of claim 2, wherein, The His tag is a lengthened tag containing 10 histidines; and the amino acid sequence of the Strep II tag is WSHPQFEKGGGSGGGSGGSAWSHPQFEK.
4. The purification method of claim 3, wherein, The eukaryotic cells in step S1 are mammalian Expi293 cells or insect SF9 cells.
5. The purification method of claim 1, wherein, The resuspension buffer used in step S2 comprises: a Tris-HCl buffer with a concentration of 100 mM, sodium chloride with a concentration of 300 mM, and glycerol with a concentration of 20%; and the detergent used in step S2 is dodecyl-beta-D-maltoside or lauryl maltose neopentyl glycol, and the concentration of the detergent is 1%-2%.
6. The purification method of claim 1, wherein, The Strep packing material in step S3 is Strep-TactinXT 4Flow.
7. The purification method of claim 6, wherein, The specific steps of affinity chromatography purification in step S3 comprise balancing, incubation, washing and elution.
8. The purification method of claim 7, wherein, The pH value of the balancing buffer and the washing buffer used in the process of affinity chromatography purification in step S3 is 7.4-8.0; and the elution buffer is formed by adding biotin with a final concentration of 25 mM to the balancing buffer.
9. The purification method of claim 1, wherein, The nickel column packing material in step S4 is NI-NTAAgarose, and the specific steps of affinity chromatography purification in step S4 comprise balancing, loading, washing and elution.
10. The purification method of claim 9, wherein, The pH value of the balancing buffer used in the process of affinity chromatography purification in step S4 is 7.4-8.0, the washing buffer is formed by adding imidazole with a final concentration of 15-30 mM to the balancing buffer, and the elution buffer is formed by adding imidazole with a final concentration of 250-500 mM to the balancing buffer.