Nanometer antibody interacting with SUMO or bdSUMO tag protein and application thereof

Abstract

The invention discloses a nano antibody interacting with SUMO or bdSUMO tag protein and application of the nano antibody. The nano antibody is S1D9-Nb, and the amino acid sequence is shown in SEQ ID NO.1. The nano antibody S1D9-Nb can be efficiently expressed in escherichia coli, the production process is simple, the cost is low, and the yield is high. The S1D9-Nb has high affinity with the SUMO protein and the bdSUMO protein, and can be used for detecting, enriching and purifying the SUMO and the bdSUMO or the fusion protein of the SUMO and the bdSUMO. In addition, the invention also provides an S1D9-Nb-SUMO compound and a protein crystal and a structure of the S1D9-Nb-bdSUMO compound, the S1D9-Nb-SUMO obtains a high-resolution structure with the resolution of 2.10, and the S1D9-Nb-bdSUMO obtains a high-resolution structure with the resolution of 1.85, so that an important structural basis is provided for understanding a molecular mechanism of SUMO / bdSUMO modification, and the S1D9-Nb-SUMO compound has good application prospects. The method has wide application prospects in the fields of protein engineering, drug research and development, biosensors and the like.

Description

Technical Field

[0001] This invention relates to the field of protein engineering technology, specifically to a nanobody that interacts with SUMO or bdSUMO-tagged proteins and its applications. Background Technology

[0002] Nanobodies, a class of single-domain antibodies first discovered in camels, are single-domain antibody fragments composed of the variable region (VHH) of a heavy chain antibody. Each single variable region of the heavy chain is sufficient to recognize and bind to an antigen, offering advantages such as small molecular weight, high affinity, and stability. Furthermore, the preparation process of nanobodies is relatively simple, allowing for large-scale production through genetic engineering techniques. In addition, nanobodies exhibit very strong specific recognition capabilities, specifically binding to specific epitopes of target molecules. Simultaneously, antibody nanobodies can also be used to separate and purify target proteins, improving protein purity and yield. The unique structural and functional properties of nanobodies make them promising candidates for a wide range of applications in the field of biotechnology.

[0003] SUMO (small ubiquitin-like modified proteins) are a class of small proteins widely found in eukaryotes. SUMO tags have a relatively small molecular weight, approximately 10 to 20 kDa, offering unique advantages in protein expression and purification. They can improve the solubility of target proteins, reduce protein aggregation, thereby increasing the yield and activity of target proteins, thus providing sufficient and high-quality material for further protein research. During protein purification, SUMO tags can achieve efficient purification of target proteins by binding to specific affinity chromatography media. bdSUMO, derived from *Brachypodium distachyon*, is a plant-derived SUMO protein. In recent years, the application of plant-derived SUMO proteins in protein expression and functional studies has gradually attracted attention. bdSUMO, as a novel SUMO protein, possesses modification mechanisms and functional characteristics similar to traditional SUMO proteins.

[0004] With the continuous development of biotechnology, higher demands are being placed on protein research and application. More efficient, specific, and convenient methods are needed to obtain and apply target proteins during protein expression and purification. SUMO and bdSUMO tags, as excellent tools for protein expression and purification, have significant research and application value when combined with other biotechnologies. Nanobodies, as a novel antibody form, possess unique structural and functional characteristics. Combining nanobodies with SUMO and bdSUMO tags can fully leverage the specific recognition capabilities of nanobodies and the advantages of SUMO and bdSUMO tags in protein expression and purification, providing a new technological approach for protein research and application. Summary of the Invention

[0005] The purpose of this invention is to address the existing technical problems by providing a nanobody that interacts with SUMO or bdSUMO-tagged proteins and its applications.

[0006] The present invention achieves the above objectives through the following technical solutions: As a first aspect of the present invention, a nanobody that interacts with a SUMO or bdSUMO-tagged protein is provided, the nanobody being S1D9-Nb, obtained by yeast surface display technology, the amino acid sequence of the S1D9-Nb being shown in SEQ ID NO.1.

[0007] As a second aspect of the invention, a gene encoding a nanobody as described above is also provided, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] As a third aspect of the present invention, a recombinant plasmid is also provided, wherein the recombinant plasmid is an expression vector containing the gene as described above, and is capable of correspondingly translating and expressing the nanobody as described above, wherein the expression vector is pET22b.

[0009] As a fourth aspect of the invention, a host cell is also provided, which is Escherichia coli BL21 transfected with the recombinant plasmid as described above, preferably Escherichia coli BL21(DE3) cells.

[0010] As a fifth aspect of the invention, an affinity medium for purifying SUMO or bdSUMO-tagged proteins is also provided, the affinity medium containing nanobodies and Strep-Tactin fillers that interact with SUMO or bdSUMO-tagged proteins as described above.

[0011] As a sixth aspect of the present invention, the use of a nanobody as described above in any of the following (1)-(2), (1) detecting interaction with SUMO or bdSUMO-tagged proteins; (2) enriching, detecting or purifying SUMO-tagged proteins, bdSUMO-tagged proteins, SUMO-tagged protein fusion proteins or bdSUMO-tagged protein fusion proteins.

[0012] As a seventh aspect of the present invention, a protein crystal S1D9-Nb-SUMO, a complex of SUMO-tagged protein and nanobody, is also provided. The crystallization conditions of the S1D9-Nb-SUMO complex protein crystal are 0.2M lithium chloride, 0.1M sodium acetate, pH=5.0, and 20% PEG6000.

[0013] As an eighth aspect of the present invention, a protein crystal S1D9-Nb-bdSUMO, a protein complex of a bdSUMO-tagged protein and a nanobody, is also provided. The crystallization conditions of the bdSUMO-nanobody complex protein crystal are 0.2M sodium chloride, 0.1M Bis-Tris (bis(2-hydroxyethyl)aminomethane), pH=5.5, and 25% PEG3350.

[0014] As a further optimization of the present invention, the S1D9-Nb-SUMO protein crystal has a resolution of 2.10 Å, cell parameters of 59.96, 139.26, 140.37, 90.00, 90.00, 90.000, and space group of P22121.

[0015] As a further optimization of the present invention, the S1D9-Nb-bdSUMO has a resolution of 1.85 Å, and the crystal cell parameters are 98.22, 36.13, 57.47, 90.00, 92.29, 90.00; the space group is C121.

[0016] The present invention has the following beneficial effects: This invention provides a nanobody S1D9-Nb that can interact with SUMO or bdSUMO-tagged proteins. The S1D9-Nb nanobody has high affinity for SUMO and bdSUMO proteins, with a specific affinity of 165 nM and 150 nM, respectively, and can be used for the detection and purification of SUMO / bdSUMO or SUMO / bdSUMO fusion proteins.

[0017] Furthermore, this invention also provides protein crystals and structures of the S1D9-Nb-SUMO complex and the S1D9-Nb-bdSUMO complex. The S1D9-Nb-SUMO complex obtained a high-resolution structure with a resolution of 2.10 Å, and the S1D9-Nb-bdSUMO complex obtained a high-resolution structure with a resolution of 1.85 Å. These findings provide an important structural basis for understanding the molecular mechanism of SUMO / bdSUMOylation modification and have broad application prospects in protein engineering, drug development, and biosensors.

[0018] Finally, the nanobody S1D9-Nb provided by this invention can be efficiently expressed in Escherichia coli, and the production process is simple, low-cost and high-yield, thus having a broad application market. Attached Figure Description

[0019] Figure 1 Image showing the affinity purification of nanobody S1D9-Nb; Figure 2 Image showing the purification process of the S1D9-Nb nanobody using molecular sieves; Figure 3 FSEC assay of the interaction between nanobody S1D9-Nb and SUMO and bdSUMO; Figure 4 Affinity assays of nanobody S1D9-Nb with SUMO and bdSUMO; Figure 5 This diagram shows the preparation of the nanobody with S1D9-Nb affinity and the medium. Figure 6 Image showing the effect of purifying protein with the nanobody S1D9-Nb; Figure 7 SEC detection maps were prepared for the S1D9-Nb-SUMO complex and the S1D9-Nb-bdSUMO complex; Figure 8 Three-dimensional structural diagrams of the S1D9-Nb-SUMO complex and the S1D9-Nb-bdSUMO complex. Detailed Implementation

[0020] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0021] 1. Materials Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art. Where specific conditions are not specified, they shall be performed according to conventional conditions or conditions recommended by the manufacturer. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0022] 2. Method 2.1 Expression of nanobody S1D9-Nb protein Nanobodies were obtained using yeast surface display technology. The yeast surface-display nanobody library was obtained from the paper "Yeast surface display platform for rapid discovery of conformationally selective nanobodies" (2018).

[0023] The specific steps are as follows: After two rounds of magnetic bead sorting (MACS) and one round of flow cytometry sorting (FACS), 131,300 yeast cells were obtained. The sorted yeast cells were plated, and 400 single clones were selected for flow cytometry analysis. 24 clones showed significant binding signals to SUMO and bdSUMO. These 24 yeast samples were amplified, plasmids were extracted, and their DNA sequences were obtained through PCR and sequencing. After removing repetitive sequences, a nanobody S1D9-Nb was obtained, hereinafter referred to as S1D9-Nb.

[0024] The amino acid sequence of S1D9-Nb is shown in SEQ ID NO.1, and the gene sequence encoding S1D9-Nb is shown in SEQ ID NO.2.

[0025] To facilitate subsequent purification of the S1D9-Nb protein, a pelB signal peptide was added to the N-terminus of the amino acid sequence of S1D9-Nb as shown in SEQ ID NO.1. The amino acid sequence of the pelB signal peptide is MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO.3). A TEV-6His-StrepII tag was added to the C-terminus, where 'TEV' represents the protease cleavage site (ENLYFQG), 6His is the HHHHHH histidine tag, and the StrepII sequence is WSHPQFEK. The amino acid sequence of the TEV-6His-StrepII tag is ENLYFQGHHHHHHWSHPQFEK (SEQ ID NO.4).

[0026] Subsequently, the sequence was constructed on pET22b (GenScript) to form the pET22b-pelB-S1D9-Nb-TEV-6His-Strep II recombinant plasmid. The recombinant plasmid was verified by sequencing to be completely consistent with the target sequence.

[0027] After obtaining the recombinant plasmid, the successfully constructed nanobody recombinant plasmid was transformed into BL21(DE3) Escherichia coli competent cells. Single colonies were picked and cultured overnight at 37°C in 50 mL LB broth. The overnight cultured bacteria were then inoculated into 1 L LB broth at a ratio of 1:100 and cultured at 37°C until the bacterial culture reached OD. 600 When the bacterial growth rate is 0.6-0.8, add 0.5mM IPTG and incubate overnight at 15℃. Collect the bacterial cells by centrifugation at 5000rpm.

[0028] 2.2 Purification of S1D9-Nb protein The collected bacterial cells were weighed and added to buffer A (50 mM Tris-HCl (pH=7.5), 300 mM NaCl, 10% glycerol) at a ratio of 1:10. The cells were homogenized using a high-pressure homogenizer, and the supernatant was collected by centrifugation at 16,000 rpm. Proteins were enriched and purified using affinity chromatography with Strep-Tactin XT. Before purification, the Strep-Tactin XT column was equilibrated with buffer A. After all cell supernatant was attached to the column, the column was washed once with buffer A, and the protein was eluted with 75 mM biotin. The eluted protein was collected for SDS-PAGE analysis.

[0029] Purification results are as follows Figure 1 As shown, the S1D9-Nb protein could be eluted with 75 mM biotin, but its purity was poor. Therefore, the eluted protein was further subjected to gel filtration chromatography (SEC) using a HiLoad 16 / 600 Superdex 75 pg column. The gel chromatography buffer consisted of 50 mM HEPES, pH 7.5, 150 mM NaCl, and 5% glycerol. The target protein was collected based on the peak chromatogram from the gel filtration chromatography, and the collected sample was concentrated and subjected to SDS-PAGE quality analysis.

[0030] The experimental results are shown in Figure 2 Peak 3 of the target protein was collected, and the purity of S1D9-Nb was significantly improved after SEC purification.

[0031] 2.3 Detection of the interaction between S1D9-Nb and SUMO and bdSUMO 2.3.1 Verification of S1D9-Nb combined with SUMO and bdSUMO To verify whether the screened S1D9-Nb can bind to SUMO and bdSUMO proteins (both purchased from Biortus) in vitro, the purified S1D9-Nb from step 2.2 was subjected to FSEC analysis with SUMO and bdSUMO proteins, respectively. The purchased SUMO and bdSUMO proteins were untagged proteins. S1D9-Nb has a 6His tag at its C-terminus, which can bind to HisLite. If S1D9-Nb binds to either SUMO or bdSUMO proteins, the protein peak will appear shifted forward on the FSEC assay. The specific procedure is as follows: The purified S1D9-Nb protein was incubated on ice for 1 hour with SUMO and bdSUMO at a molar ratio of 1.1:1, centrifuged at 12,000 rpm for 1 hour, and then detected by FSEC. A separate S1D9-Nb protein sample was used as a control.

[0032] The experimental results are shown in Figure 3Peak 1 is S1D9-Nb, and peak 2 is the S1D9-Nb+SUMO or S1D9-Nb+bdSUMO complex peak. Peak 2 shows a significant forward migration compared to peak 1, indicating that the nanobody S1D9-Nb protein can bind to SUMO or bdSUMO protein in vitro.

[0033] 2.4. Detection of S1D9-Nb affinity with SUMO and bdSUMO To further clarify the affinity of the nanobody S1D9-Nb for SUMO and bdSUMO, respectively, the SPR method was used to detect the affinity of the nanobody S1D9-Nb for SUMO and bdSUMO. The specific procedures are as follows: The concentration of the nanobody S1D9-Nb was diluted to 10 μg / ml using running buffer (10 mM Hepes (pH=7.5), 150 mM NaCl, 0.05% Tween 20). The CM5 chip was activated with NHS (N-hydroxysuccinimide) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbazolamine). S1D9-Nb was added to the CM5 chip at a flow rate of 5 μl / min for a total of 410 s. Blocking was performed using 1 M ethanolamine salt at a flow rate of 10 μl / min for a total of 420 s. Then, different concentrations of SUMO or bdSUMO protein were added at a flow rate of 30 μl / min, with a binding time of 90 s and a dissociation time of 1800 s. After the program ran, data analysis was performed using software.

[0034] The experimental results are shown in Figure 4 According to the analyzed data, S1D9-Nb has high affinity for SUMO and bdSUMO, at 165 nM and 150 nM, respectively.

[0035] 2.4 Application of S1D9-Nb in protein purification 2.4.1 Preparation of S1D9-Nb affinity mediator To verify whether S1D9-Nb can be used for the purification of SUMO or bdSUMO-tagged proteins, taking the SUMO tag as an example, S1D9-Nb was prepared into an affinity medium for purification and detection. The specific preparation process is as follows: The C-terminus of the S1D9-Nb protein contains a StrepII sequence. The StrepII tag can bind to Strep-Tactin packing material. Therefore, 50 μl of Strep-Tactin packing material was taken, and the Strep-Tactin column was first equilibrated with buffer B (25 mM HEPES, pH 7.5, 500 mM NaCl, 1 mM TCEP). Then, excess S1D9-Nb was added to the packing material. After incubation at 4°C for 1 hour, the column was centrifuged at 12000 rpm for 2 min. 1 ml of buffer B was added to the packing material for washing. After washing, the column was centrifuged at 12000 rpm for 2 min. This process was repeated 3 times. The supernatant from the last centrifugation was used for SDS-PAGE analysis. In addition, after the last centrifugation, 20 μl of buffer B was added to the packing material, and 2 μl of the packing material was used for SDS-PAGE analysis to determine whether the S1D9-Nb affinity medium was successfully prepared.

[0036] The experimental results are shown in Figure 5 The fact that nanobodies could not be detected in the buffer-washed sample (final supernatant) but could be detected in the filler sample indicates that the S1D9-Nb affinity medium matrix has been successfully prepared and that the amount of S1D9-Nb immobilized in the matrix has been saturated.

[0037] 2.4.2 Application of S1D9-Nb affinity mediator in protein purification After preparing the S1D9-Nb affinity medium, to verify its suitability for purifying SUMO-tagged proteins, three SUMO-tagged proteins (target protein 1, target protein 2, and target protein 3) were tested. The sequences are shown in SEQ ID Nos. 5-7, and the SUMO and bdSUMO tag sequences are shown in SEQ ID Nos. 8-9, respectively. The specific procedures are as follows: Add the test proteins (target protein 1, target protein 2, and target protein 3) tagged with SUMO to the S1D9-Nb affinity medium and incubate at 4°C for 1 hour. Centrifuge at 12000 rpm for 5 min and collect the supernatant (referred to as the breakthrough sample) to prepare the SDS-PAGE assay sample. Then, add 1 ml of buffer B to the packing material for washing. After washing, centrifuge at 12000 rpm for 5 min, repeating this process 3 times. Collect the supernatant from the last wash (referred to as the washing sample) to prepare the SDS-PAGE assay sample. Add 20 μl of buffer B to the packing material and use 2 μl of packing material to prepare the SDS-PAGE assay sample.

[0038] The experimental results are shown in Figure 6 SUMO-tagged proteins could not be detected in either the penetration or washing samples, but S1D9-Nb, target protein 1, target protein 2, and target protein 3 could be detected in the packing material, indicating that the three SUMO-tagged target proteins were basically enriched by the S1D9-Nb affinity medium.

[0039] 2.5 Crystallization and structural analysis of S1D9-Nb-SUMO and S1D9-Nb-bdSUMO complexes 2.5.1 Preparation of S1D9-Nb-SUMO and S1D9-Nb-bdSUMO complexes The S1D9-Nb purified in step 2.2 was incubated with SUMO and bdSUMO proteins at an equimolar ratio at room temperature for 1 hour, followed by SEC purification. SEC results are shown below. Figure 7 The experimental results showed that after incubation of S1D9-Nb with SUMO protein, a single peak Peak1 appeared. SDS-PAGE analysis showed that Peak1 was a complex. After incubation of S1D9-Nb with bdSUMO protein, a single peak Part1 appeared. SDS-PAGE analysis showed that Part1 was a complex. The protein peaks of S1D9-Nb-SUMO and S1D9-Nb-bdSUMO complexes were collected for the next crystallization experiment.

[0040] 2.5.2 Crystallization of S1D9-Nb-SUMO and S1D9-Nb-bdSUMO complexes The S1D9-Nb-SUMO and S1D9-Nb-bdSUMO obtained above were crystallized using the sitting drop method, respectively. The PEG Kit and Index Kit from Hampton Research, and the Morpheus II and Morpheus III Kits and Complexes from QIAGEN were selected for crystallization screening. The specific procedures were as follows: Add 15 μL of crystallization reagent to a 96-well crystallization plate as a buffer solution. Spot the sample using a Mosquito LCP protein crystallization screener, adding 200 nm of protein to 200 nm of crystallization solution. Seal the 96-well plate with the MicroAmp Optical Adhesive membrane and incubate at 20°C. Observe crystal growth periodically.

[0041] Crystallization screening of the S1D9-Nb-SUMO and S1D9-Nb-bdSUMO complexes was conducted. The S1D9-Nb-SUMO complex yielded crystals in approximately 11 days at a protein concentration of 9.77 mg / ml, with crystallization conditions of 0.2 M lithium chloride, 0.1 M sodium acetate, pH 5.0, and 20% PEG6000. The S1D9-Nb-bdSUMO complex yielded relatively good crystals in approximately 20 days at a protein concentration of 12.61 mg / ml, with crystallization conditions of 0.2 M sodium chloride, 0.1 M Bis-Tris (bis(2-hydroxyethyl)aminomethane), pH 5.5, and 25% PEG3350.

[0042] 2.5.3 Structural analysis of S1D9-Nb-SUMO and S1D9-Nb-bdSUMO complexes The S1D9-Nb-SUMO and S1D9-Nb-bdSUMO composite crystals obtained above were subjected to X-ray diffraction at the Japanese synchrotron radiation source SPring-8. The unit parameters of the S1D9-Nb-SUMO composite crystal were 59.96, 139.26, 140.37, 90.00, 90.00, and 90.000; the space group was P22121, and the composite structure was finally resolved with a resolution of 2.1 Å. The unit parameters of the S1D9-Nb-bdSUMO composite crystal were 98.22, 36.13, 57.47, 90.00, 92.29, and 90.00; the space group was C121, and the high-resolution structure was finally resolved with a resolution of 1.85 Å. The overall structural diagrams of the two composites are shown below. Figure 8 .

[0043] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A nanobody that interacts with a SUMO or bdSUMO-tagged protein, characterized in that, The nanobody is S1D9-Nb, and the amino acid sequence of S1D9-Nb is shown in SEQ ID NO.

1.

2. The gene encoding the nanobody as described in claim 1, characterized in that, The nucleotide sequence of the gene encoding the nanobody is shown in SEQ ID NO.

2.

3. A recombinant plasmid, characterized in that, The recombinant plasmid is an expression vector containing the gene as described in claim 3, and is capable of correspondingly translating and expressing the nanobody as described in claim 1, wherein the expression vector is pET22b.

4. A host cell, characterized in that, Escherichia coli BL21 that has been transformed into the recombinant plasmid as described in claim 3.

5. An affinity medium for purifying SUMO or bdSUMO-tagged proteins, characterized in that, The affinity medium contains the nanobody and Strep-Tactin filler that interact with the SUMO or bdSUMO-tagged protein as described in claim 1.

6. The use of a nanobody as described in claim 1 in any one of the following (1)-(2), characterized in that, (1) Detection of interactions with SUMO or bdSUMO-tagged proteins; (2) Enrichment, detection or purification of SUMO-tagged proteins, bdSUMO-tagged proteins, SUMO-tagged protein fusion proteins or bdSUMO-tagged protein fusion proteins.

7. A protein crystal S1D9-Nb-SUMO, a complex of SUMO-tagged protein and nanobody, characterized in that... The crystallization conditions for the S1D9-Nb-SUMO complex protein crystals were 0.2M lithium chloride, 0.1M sodium acetate, pH=5.0, and 20% PEG6000.

8. A protein crystal S1D9-Nb-bdSUMO, a complex of bdSUMO-tagged protein and nanobody, characterized in that... The crystallization conditions for the bdSUMO-nano antibody complex protein crystals were 0.2M sodium chloride, 0.1M Bis-Tris, pH=5.5, and 25% PEG3350.

9. The SUMO-tagged protein and nanobody complex protein crystal S1D9-Nb-SUMO according to claim 7, characterized in that, The S1D9-Nb-SUMO protein crystal has a resolution of 2.10 Å, cell parameters of 59.96, 139.26, 140.37, 90.00, 90.00, and 90.000, and a space group of P22121.

10. The bdSUMO-tagged protein and nanobody complex protein crystal S1D9-Nb-bdSUMO according to claim 8, characterized in that, The S1D9-Nb-bdSUMO crystal has a resolution of 1.85 Å, and its cell parameters are 98.22, 36.13, 57.47, 90.00, 92.29, and 90.00; its space group is C121.

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