An anti-eGFP nanobody matrix, preparation method and application thereof

Abstract

The application discloses an Anti-eGFP nanobody matrix and a preparation method and application thereof, relates to the technical field of bioengineering, and the Anti-eGFP nanobody matrix comprises an affinity purification matrix and an adapting solution; wherein the affinity purification matrix comprises an immunoprotein ImS2 of pseudomonas aeruginosa, and the amino acid sequence is shown as SEQ ID NO. 5; the adapting solution is a fusion protein InAS2-Anti-eGFP nanobody of high-affinity protein InAS2 corresponding to the immunoprotein ImS2 of pseudomonas aeruginosa and Anti-eGFP nanobody, and the amino acid sequence is shown as SEQ ID NO. 1. The matrix has the characteristics of high affinity, strong specificity and high loading capacity, and the purification effect is equivalent to that of a commercially available high-affinity purification column Strep-Tactin XT; the purified protein can be eluted by enzyme cutting, the purity of the purified protein is high, in addition, the new matrix can be repeatedly used, the use cost can be reduced, and the new matrix has better application value in protein separation and purification.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to an Anti-eGFP nanobody matrix, its preparation method, and its application. Background Technology

[0002] Green fluorescent protein (GFP) is a protein composed of approximately 238 amino acids that emits fluorescence in the green portion of the visible spectrum. GFP is stable under various harsh conditions, has a small molecular weight, and exhibits no cytotoxicity when expressed at high levels, making it a promising candidate for applications in modern cell biology and molecular biology research. Enhanced green fluorescent protein (EGFP) is a mutant strain of GFP that emits fluorescence with an intensity more than six times greater than GFP and exhibits even better photostability. EGFP demonstrates significant performance improvements over traditional GFP, making it a more popular choice in various biological research fields.

[0003] Nanobodies, also known as single-domain antibodies, are unique antibody fragments derived from camels and sharks. They consist of a single antibody-binding domain (VHH) and can specifically bind to target antigens. Nanobodies possess advantages such as small molecular weight, simple and easily foldable structure, high stability, and high specificity and affinity, making them a popular choice for drug development and biological applications.

[0004] Leveraging the advantages of both, researchers have designed anti-eGFP nanobodies specifically for green fluorescent protein (GFP), which can be used in various scientific applications. Among these, anti-eGFP nanobody affinity chromatography matrices targeting both GFP and EGFP can be used for the affinity purification of fusion proteins tagged with GFP or EGFP. However, existing conventional anti-eGFP nanobody affinity chromatography matrices mostly suffer from limitations such as low loading capacity, non-reusability, and high cost. Therefore, researching new anti-eGFP nanobody affinity chromatography matrices to overcome these limitations is of significant research importance in improving their application value in protein separation and purification. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an Anti-eGFP nanobody matrix, its preparation method, and its application.

[0006] The present invention achieves the above objectives through the following technical solutions:

[0007] As a first aspect of the present invention, an Anti-eGFP nanobody matrix is ​​provided, comprising an affinity purification matrix and an adaptor solution; wherein the affinity purification matrix comprises Pseudomonas aeruginosa immune protein ImS2, the amino acid sequence of which is shown in SEQ ID NO.5;

[0008] The adaptor solution is an InAS2-Anti-eGFP nanobody, a fusion protein of InAS2, a high-affinity protein corresponding to the Pseudomonas aeruginosa immunoprotein ImS2, and an Anti-eGFP nanobody, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] A further improvement is that the nucleotide sequence encoding the InAS2-Anti-eGFP nanobody is shown in SEQ ID NO.2.

[0010] A further improvement is that the vector expressing the recombinant Anti-eGFP nanobody fusion protein is pET-28a, and the strain expressing the recombinant Anti-eGFP nanobody fusion protein is Escherichia coli BL21(DE3).

[0011] As a second aspect of the present invention, a method for preparing an Anti-eGFP nanobody matrix is ​​also provided. The method involves first immobilizing the Pseudomonas aeruginosa immune protein ImS2 onto an affinity matrix to obtain an affinity purified matrix, and then loading the InAS2-Anti-eGFP nanobody onto the affinity purified matrix.

[0012] As a third aspect of the present invention, a method for protein purification using the Anti-eGFP nanobody matrix as described in any of the above-described methods is also provided, comprising the following steps:

[0013] (1) The recombinant target protein with GFP or EGFP tag and protease cleavage site fused to the N-terminus or C-terminus to be purified is expressed in a heterologous expression cell line.

[0014] (2) Harvest the cells containing the target protein to be purified, and lyse the cells to obtain cell supernatant containing the target protein fused with GFP or EGFP tag and protease cleavage site;

[0015] (3) Incubate the cell supernatant with the Anti-eGFP nanobody matrix to enrich the target protein fused with GFP or EGFP tags and protease cleavage sites;

[0016] (4) Wash the Anti-eGFP nanobody matrix with a buffer containing the protease corresponding to the restriction site. After the GFP or EGFP tag is removed, the target protein flows down and the purified target protein can be obtained.

[0017] A further improvement is that the protein purification method further includes regenerating the affinity purification matrix using 6M guanidine hydrochloride or 8M urea.

[0018] The novel Anti-eGFP nanobody matrix developed in this invention comprises two parts: an affinity purification matrix and an adaptation solution. The affinity purification matrix is ​​a reusable matrix prepared based on the Pseudomonas aeruginosa immune protein ImS2, and the adaptation solution is a fusion protein of a high-affinity protein that can interact with the Pseudomonas aeruginosa immune protein ImS2 and the Anti-eGFP nanobody, namely InAS2-Anti-eGFP nanobody.

[0019] The principle of using this new Anti-eGFP nanobody matrix for protein purification is as follows: the target protein containing a GFP or EGFP tag and a protease cleavage site will bind to the new packing material because it interacts with the eGFP nanobody. Then, it is eluted by enzyme digestion with a buffer containing a protease that can recognize the corresponding cleavage site. The target protein after enzyme digestion does not have a GFP or EGFP tag and will not bind to the matrix, so it will flow through. Therefore, the purified target protein can be obtained by collecting the flow-through.

[0020] This new matrix is ​​reusable. After washing with 6M guanidine hydrochloride (GdnHCl) or 8M urea, the InAS2-Anti-eGFP nanobody protein denatures and detaches from the ImS2 matrix, which is then regenerated. After multiple washes with buffer, the ImS2 matrix can be directly loaded with fresh InAS2-Anti-eGFP nanobody to form a new ImS2-InAS2-Anti-eGFP nanobody matrix. This matrix can then be used to purify new GFP or EGFP-tagged fusion proteins. The entire process only requires replacing the adaptor solution (InAS2-Anti-eGFP nanobody).

[0021] In summary, the present invention has the following beneficial effects:

[0022] The novel Anti-eGFP nanobody matrix developed in this invention features high affinity, strong specificity, and high loading capacity. It can enrich recombinant proteins fused with GFP or EGFP tags, increasing the loading capacity by approximately 7 times compared to existing conventional Anti-eGFP nanobody matrices, significantly reducing matrix costs. Furthermore, its purification effect is comparable to that of the commercially available high-affinity purification column Strep-Tactin XT. Proteins can also be eluted by enzyme digestion, resulting in high-purity proteins. In addition, this new matrix is ​​reusable, greatly reducing usage costs and demonstrating greater application value in protein separation and purification. Attached Figure Description

[0023] Figure 1 The purification results are for the InAS2-Anti-eGFP nanobody protein.

[0024] Figure 2 The purification results of Anti-eGFP nanobody protein are shown in the control image.

[0025] Figure 3 The purification results of ImS2 protein;

[0026] Figure 4 The results show the purification of EGFP protein;

[0027] Figure 5 The results show the affinity assay between EGFP protein and InAS2-Anti-eGFP nanobody protein.

[0028] Figure 6 The loading results are for the Anti-eGFP nanobody matrix.

[0029] Figure 7 The results of purification tests on protein A using ImS2-InAS2-Anti-eGFP nanobody;

[0030] Figure 8 The results of purification tests on protein B using ImS2-InAS2-Anti-eGFP nanobody;

[0031] Figure 9 The results show the further purification of protein A after enzyme digestion;

[0032] Figure 10 The results show the further purification of protein B after enzyme digestion. Detailed Implementation

[0033] 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.

[0034] 1. Materials and Reagents

[0035] Unless otherwise specified, the methods used in this embodiment are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products.

[0036] 2. Method

[0037] 2.1 Plasmid Construction

[0038] 2.1.1 Gene Synthesis of Anti-eGFP Nanobody Protein

[0039] The gene sequence of the Anti-eGFP nanobody protein was obtained through gene synthesis. The protein sequence of InAS2-Anti-eGFP nanobody is shown in SEQ ID NO.1, and the gene sequence of InAS2-Anti-eGFP nanobody is shown in SEQ ID NO.2. To improve protein yield, an 8His-SUMO tag was added to the N-terminus of the gene sequence; the tag sequence is shown in SEQ ID NO.3 (8His is the affinity tag, and SUMO is the SUMO protease recognition tag sequence).

[0040] The gene sequence of the synthesized 8His-SUMO-InAS2-Anti-eGFP nanobody was constructed on the pET-28a vector, and all sequences were verified to be correct by the sequencing company.

[0041] In addition, a control Anti-eGFP nanobody plasmid was synthesized to prepare a common Anti-eGFP nanobody matrix for comparison with the newly purified matrix. The sequence of the control Anti-eGFP nanobody is shown in SEQ ID NO.4. It was constructed on the pGEX6P-1 vector, which has a built-in GST tag and PreScission restriction site for protein purification.

[0042] 2.1.2 Gene Synthesis of Pseudomonas aeruginosa ImS2 Immunoprotein

[0043] The *Pseudomonas aeruginosa* immunoprotein ImS2 used in this embodiment, with its amino acid sequence shown in SEQ ID NO. 5, is derived from the wild-type ImS2 protein by adding the amino acid "CGG" to its N-terminus. A 6His tag and a 3C restriction enzyme site (6His tag sequence is HHHHHH, and the 3C restriction enzyme site sequence is LEVLFQGP) were added to the N-terminus of the *Pseudomonas aeruginosa* immunoprotein ImS2 sequence shown in SEQ ID NO. 5, and the protein was constructed in the pET-28a vector for protein purification. All genes encoding this immunoprotein were synthesized, and all synthesized genes were verified by a sequencing company.

[0044] 2.2 Protein Expression

[0045] 2.2.1 Expression of InAS2-Anti-eGFP nanobody protein

[0046] The plasmid of the correctly sequenced synthesized InAS2-Anti-eGFP nanobody protein was transformed into competent BL21(DE3) cells and cultured overnight at 37°C. Single colonies were picked and inoculated into 5 mL of LB broth and cultured overnight at 37°C. The overnight culture was then inoculated into 1 L of LB broth at a ratio of 1:100 to express the protein at high concentrations, 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.

[0047] 2.2.2 Expression of Pseudomonas aeruginosa immunoprotein ImS2

[0048] The expression method for the immune protein ImS2 is the same as that for the expression of InAS2-Anti-eGFP nanobody protein in step 2.2.1, and the volume for large-scale expression is 1L.

[0049] 2.3 Protein purification

[0050] 2.3.1 Purification of InAS2-Anti-eGFP nanobody protein

[0051] The bacterial cells expressed in step 2.2.1 were collected and weighed. Lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM imidazole) was added at a 1:10 ratio, and the cells were homogenized using a high-pressure homogenizer. The supernatant was collected by centrifugation at 16,000 rpm. The Anti-eGFP nanobody protein in the novel matrix design carries a His tag. The protein was enriched and purified using a Ni Bestarose FF affinity chromatography column. The specific procedure was as follows: first, the Ni Bestarose FF affinity chromatography column was equilibrated with 10 column volumes of lysis buffer. Then, the lysis supernatant was loaded onto the Ni Bestarose FF affinity chromatography column and eluted with imidazole solution. The protein eluted with 300 mM imidazole was collected. For further application, the 8-His-SUMO tag was removed. SUMO enzyme was added to the collected target protein solution, and the protein was digested overnight at 4°C. The digested protein was then reloaded onto a Ni Bestarose FF column. Proteins without a His tag did not bind to the column, so the flow-through protein was collected. The protein purification results are as follows: Figure 1 A high-purity InAS2-Anti-eGFP nanobody protein was obtained. The final yield of InAS2-Anti-eGFP nanobody protein was 64.8 mg / L.

[0052] The purification procedure for the anti-eGFP nanobody can be referenced from the above steps. The lysis buffer consisted of 50 mM M HEPES pH 7.5, 500 mM NaCl, 5% glycerol, and 20 mM imidazole. GST FF column affinity chromatography was used, followed by PreScission protease digestion. After digestion, a reverse affinity chromatography was performed using a GST FF column, and the flow-through target protein was collected. The final protein purification results are shown below. Figure 2 We obtained a highly pure, unlabeled Anti-eGFP nanobody protein, which will be used to prepare a control Anti-eGFP nanobody filler.

[0053] 2.3.2 Purification of Pseudomonas aeruginosa immunoprotein ImS2

[0054] After high-level expression of the immunoprotein ImS2 in step 2.2.2, the collected bacterial cells were weighed and added to a 1:10 ratio with the appropriate volume of lysis buffer (50mM Tris-HCl pH 7.5, 500mM NaCl, 5% glycerol). The cells were homogenized using a high-pressure homogenizer, and the supernatant was collected by centrifugation at 16000 rpm. Since the immunoprotein ImS2 contains a His tag, purification was performed using a Ni Bestarose FF affinity chromatography column. The specific procedure was as follows: first, the Ni Bestarose FF affinity chromatography column was washed and equilibrated with lysis buffer to 10 column volumes. Then, the lysis supernatant was loaded onto the Ni Bestarose FF affinity chromatography column, and eluted with imidazole solutions of different gradients. The protein eluted with 300mM imidazole was collected. 3C enzyme was added, and the protein was digested overnight at 4°C. The digested protein was then loaded back onto the Ni Bestarose FF column to remove the tag. Since the protein without the His tag did not bind to the column, the flow-through protein was collected. The purification results of ImS2 protein are as follows: Figure 3 A high-purity ImS2 protein was obtained. The obtained ImS2 protein will be used to prepare a new modified Anti-eGFP nanobody matrix.

[0055] 2.4 EGFP protein expression and purification

[0056] This invention constructs and expresses a 6His-TEV-GST-3C-EGFP fusion protein, which is then purified for testing the affinity between the Anti-eGFP nanobody and the EGFP protein, as well as for determining the loading capacity of the Anti-eGFP nanobody matrix. The EGFP protein sequence is shown in SEQ ID NO. 6, with a 6His-TEV-GST-3C tag sequence added to the N-terminus. The 6His-TEV-GST-3C tag sequence is shown in SEQ ID NO. 7. The synthesized gene sequence is constructed in the pET-28a vector, and all synthesized genes have been verified by a sequencing company. The expression method of this protein is the same as described in step 2.2.

[0057] The expressed bacterial cells were weighed and added to a buffer solution (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% glycerol) at a 1:10 ratio. The cells were then homogenized using a high-pressure homogenizer, and the supernatant was collected by centrifugation at 16,000 rpm for later use. The specific purification procedure for EGFP protein was as follows: First, the Ni Bestarose FF affinity chromatography column was washed and equilibrated with lysis buffer to 10 column volumes. Then, the lysis supernatant was loaded onto the Ni Bestarose FF affinity chromatography column and eluted with imidazole solutions of different gradients. The protein eluted with 300 mM imidazole was collected. For further application, the 6His-TEV-GST-3C tag needed to be removed. Add 3C enzyme to the collected target protein solution and incubate overnight at 4°C. Load the digested protein onto GST Bestarose FF and Ni Bestarose FF columns to remove the tag and enzyme. Proteins without GST and His tags will not bind to the column. Collect the flow-through target protein. The final protein purification result is as follows: Figure 4 High-purity EGFP protein was obtained.

[0058] 2.5 Verification of the affinity between Anti-eGFP nanobody protein and EGFP protein

[0059] The affinity of InAS2-Anti-eGFP nanobody protein for EGFP protein was verified using the SPR method. The specific procedure was as follows: InAS2-Anti-eGFP nanobody protein was diluted to 20 μg / ml with 10 mM sodium acetate at pH 5.0. NHS (N-hydroxysuccinimide) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbazolamine) were flowed through a CM5 chip at a flow rate of 10 μl / min for 600 s to activate the carboxyglucan matrix on the chip surface. The flow rate was then set to 5 μl / min for a total of 528 s to immobilize the InAS2-Anti-eGFP nanobody protein onto the activated CM5 chip. Blocking was performed with 1 M ethanolamine salt at a flow rate of 10 μl / min for a total time of 600 s. Then, different dilutions of EGFP protein were injected 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 analysis software. Experimental results are shown below. Figure 5 According to the analyzed data, the InAS2-Anti-eGFP nanobod y protein has an affinity of 6.35pM for EGFP, which is very strong and can be used for the development of subsequent matrices.

[0060] 2.6 Preparation of Anti-eGFP nanobody matrix

[0061] The purified Pseudomonas aeruginosa immunoprotein ImS2 protein from step 2.3.2 was immobilized onto a high-affinity iodine acetyl resin to obtain an ImS2 affinity matrix. Then, the purified InAS2-Anti-eGFP nanobody from step 2.3.1 was loaded onto the ImS2 affinity matrix to obtain an ImS2-InAS2-Anti-eGFP nanobody.

[0062] The specific method is as follows: Iodine acetyl resin (Genscript, L00403) was added to a 1 ml gel column and washed three times with buffer (50 mM HEPES pH 7.5, 200 mM NaCl, 5% glycerol, 1 mM TCEP), three column volumes each time. Immunoprotein ImS2 was loaded into the resin, and the flow rate was set to low. Loading was carried out overnight at 4°C. Then, the column was washed six times with the same buffer, three column volumes each time. A buffer containing 50 mM cysteine ​​was added to the column and incubated at 4°C for 2 hours to block the iodine acetyl resin. Finally, the column was washed six more times with the same buffer to obtain the ImS2 protein immunopurification matrix (ImS2 affinity column). Equilibrate 10 column volumes of 1 mL ImS2 affinity column with buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 5% glycerol, 2 mM DTT). Then load the purified InAS2-Anti-eGFP nanobody from step 2.3.1 onto the corresponding ImS2 affinity column and wash the column with buffer.

[0063] 2.6.2 Preparation of the control Anti-eGFP nanobody matrix

[0064] To further compare the purification effect of ImS2-InAS2-Anti-eGFP nanobody with that of commonly used Anti-eGFP nanobody, this invention also prepared a control Anti-eGFP nanobody matrix for comparison with the new purified matrix. The preparation method of the control Anti-eGFP nanobody matrix is ​​as follows:

[0065] Pretreatment was performed on NHS Purose 4Fast Flow resin (Qianchun, A51801-07): 10 column volumes were washed with 1 mM HCl aqueous solution, followed by 10 column volumes of pure water, and then 10 column volumes of coupling buffer (100 mM NaHCO3 pH 8.2, 500 mM NaCl). The purified control Anti-eGFP nanobody protein from step 2.3.1 was mixed with the pretreated resin and incubated overnight at 4°C. After incubation, 1 column volume was washed with blocking buffer (50 mM NaHCO3 pH 8.2, 150 mM NaCl, 50 mM Glycine), and the column was filled with blocking buffer. The mixture was incubated at 4°C for 1 hour. After incubation, 10 column volumes were washed with blocking buffer, and finally 10 column volumes were washed with washing buffer (10 mM sodium phosphate pH 7.0, 200 mM NaCl) to obtain the control Anti-eGFP nanobody matrix.

[0066] 2.7 Anti-eGFP nanobody matrix loading test

[0067] The specific procedure for the loading test is as follows: The purified EGFP protein from step 2.4 was loaded into 1 mL of prepared ImS2-InAS2-Anti-eGFP nanobody and control Anti-eGFP nanobody matrices, respectively, according to different protein amounts (mg). The equilibration buffer consisted of 50 mM Tris-HCl (pH 8.0) and 500 mM NaCl. The flow-through was collected, and the color changes of the matrix and the collected flow-through were observed after adding different amounts of EGFP protein. The Anti-eGFP nanobody matrix gradually turned into a distinct fluorescent green due to the enrichment of EGFP protein. At the same time, if the amount of EGFP protein loaded exceeded the loading capacity of the matrix in the column, it would flow through, and the flow-through would gradually show a fluorescent green color. Therefore, the amount of EGFP protein loaded in each matrix when the final flow-through began to show green was recorded as the loading capacity of each Anti-eGFP nanobody per mL. This loading information can provide specific information on how much matrix is ​​needed for subsequent protein purification.

[0068] Test results are available Figure 6 The experimental results show that, under 1 mL matrix conditions, the control Anti-eGFP nanobody matrix showed green fluorescence in the flow-through solution when 4 mg of EGFP protein was loaded, indicating a loading capacity of approximately 4 mg / mL, comparable to some commercially available matrices (https: / / abclonal.com.cn / catalog / AE074). The ImS2-InAS2-Anti-eGFP nanobody matrix only showed some green fluorescence in the flow-through solution when loaded with approximately 28 mg of EGFP, indicating a loading capacity of approximately 28 mg / mL. Therefore, comparing the loading capacities, under 1 mL matrix conditions, the modified ImS2-InAS2-Anti-eGFP nanobody matrix showed approximately 7 times higher loading capacity than the control Anti-eGFP nanobody matrix.

[0069] 2.8 Application of Anti-eGFP nanobody matrix in protein purification

[0070] To further verify the purification effect of the modified new Anti-eGFP nanobody matrix and compare it with the effect of commercially available high affinity purification columns, this invention compares the affinity column of ImS2-InAS2-Anti-eGFP nanobody matrix with the Strep-Tactin XT affinity column, and tests the purification effect of proteins A and B respectively. The amino acid sequences of proteins A and B are shown in SEQ ID NO. 8-9. Both proteins A and B were additionally tagged with a Strep II-EGFP-SUMO-GGG-6His tag at their N-terminus. The amino acid sequence of the tag is shown in SEQ ID NO. 10. Strep II, EGFP, and 6His tags are affinity tags, used to simultaneously compare the purification effects of the Strep-Tactin XT affinity column and the modified ImS2-InAS2-Anti-eGFP nanobody affinity column. SUMO is the SUMO protease recognition sequence used to cleave the Strep II-eGFP tag. The fusion proteins used for testing were constructed in the pET-28a vector. All recombinant plasmids were synthesized and verified to be correct by sequencing.

[0071] The expression methods for fusion proteins A and B were similar to those described in section 2.2, with an expression volume of 1 L. After overnight culture, bacteria were collected and weighed. Buffer solutions (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% glycerol, 2 mM DTT) were added at a 1:10 ratio. The cells were homogenized using an autoclave, and the supernatant was collected by centrifugation at 16000 rpm. The collected supernatant was divided into two equal portions and purified using a Strep-Tactin XT affinity column and an ImS2-InAS2-Anti-eGFP nanobody affinity column, respectively. The specific purification steps for each affinity column are as follows:

[0072] (1) Strep-Tactin XT affinity column

[0073] First, wash the Strep-Tactin XT affinity column with buffer for 10 column volumes. Then, load the lysis supernatant onto the column and elute with buffer containing 75 mM biotin. Collect the eluent and add SUMO enzyme for overnight digestion. Samples are then taken for SDS-PAGE detection.

[0074] (2) ImS2-InAS2-Anti-eGFP nanobody affinity column

[0075] First, wash the ImS2-InAS2-Anti-eGFP nanobody affinity column with buffer for 10 column volumes. Then, load the lysis supernatant onto the column, perform enzyme digestion and elution with buffer containing SUMO enzyme, collect the eluent and sample it for SDS-PAGE detection.

[0076] The purification effects of the two affinity columns are shown in [link to documentation]. Figure 7 and Figure 8 Proteins A and B can be purified using both the Strep-Tactin XT affinity column and the ImS2-InAS2-Anti-eGFP nanobody affinity column, yielding highly purified target proteins after enzyme digestion. Specifically, the yield of protein A purified using the Strep-Tactin XT column was 16.98 mg / L, while the yield using the ImS2-InAS2-Anti-eGFP nanobody column was 16.45 mg / L, indicating comparable yields for protein A when purified using both columns. For protein B, the yield using the ImS2-InAS2-Anti-eGFP nanobody column was 29.20 mg / L, while the yield using the Strep-Tactin XT column was 11.10 mg / L, showing a higher yield for protein B when purified using the ImS2-Anti-eGFP nanobody column than when purified using the Strep-Tactin XT column. Therefore, the purification effect of the ImS2-InAS2-Anti-eGFP nanobody affinity column is comparable to or even better than that of the Strep-Tactin XT affinity column. Proteins A and B collected after ImS2-InAS2-Anti-eGFP nanobody affinity chromatography contain enzymes, which can be removed through further purification. In this invention, the enzymes can be removed using a Ni Bestarose FF affinity chromatography column, as shown in the results. Figure 9 and Figure 10 High-purity proteins A and B were obtained.

[0077] III. Conclusion

[0078] This invention develops a novel Anti-GFP nanobody matrix, comprising two parts: a high-affinity reversible matrix and an "adaptation solution." The high-affinity reversible matrix is ​​a reusable matrix prepared based on the immunoprotein I mS2. The adaptation solution is a fusion protein of an affinity protein that interacts with the immunoprotein and the Anti-GFP nanobody, namely InAS2-Anti-eGFP nanobody. This matrix exhibits high affinity and specificity, effectively enriching recombinant proteins fused with GFP or EGFP tags. Its loading capacity is approximately 7 times higher than that of conventional Anti-eGFP nanobody matrices, significantly reducing matrix costs. Furthermore, its purification effect is comparable to that of the commercially available high-affinity purification column Strep-TactinXT. Proteins can also be eluted by enzyme digestion, resulting in high-purity purified proteins. In addition, this novel matrix is ​​reusable, greatly reducing usage costs and demonstrating greater application value in protein separation and purification.

[0079] 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. An Anti-eGFP nanobody matrix, characterized in that, The invention includes an affinity purification matrix and a suitable solution, wherein the affinity purification matrix includes Pseudomonas aeruginosa immune protein ImS2, the amino acid sequence of which is shown in SEQ ID NO.5; The adaptor solution is an InAS2-Anti-eGFP nanobody, a fusion protein of InAS2, a high-affinity protein corresponding to the Pseudomonas aeruginosa immunoprotein ImS2, and an Anti-eGF P nanobody, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The Anti-eGFP nanobody matrix according to claim 1, characterized in that, The nucleotide sequence encoding the InAS2-Anti-eGFP nanobody is shown in SEQ ID NO.

2.

3. The Anti-eGFP nanobody matrix according to claim 1, characterized in that, The vector expressing InAS2-Anti-eGFP nanobody was pET-28a, and the strain expressing InAS2-Anti-eGFP nanobody was Escherichia coli BL21(DE3).

4. A method for preparing the Anti-eGFP nanobody matrix as described in any one of claims 1-3, characterized in that, The preparation method involves first immobilizing the Pseudomonas aeruginosa immunoprotein ImS2 onto an affinity matrix to obtain an affinity purification matrix, and then loading the InAS2-Anti-eGFP nanobody onto the affinity purification matrix.

5. A method for protein purification using the Anti-eGFP nanobody matrix as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) The recombinant target protein with GFP or EGFP tag and protease cleavage site fused to the N-terminus or C-terminus to be purified is expressed in a heterologous expression cell line. (2) Harvest the cells containing the target protein to be purified, and lyse the cells to obtain cell supernatant containing the target protein fused with GFP or EGFP tag and protease cleavage site; (3) Incubate the cell supernatant with the Anti-eGFP nanobody matrix to enrich the target protein fused with GFP or EGFP tags and protease cleavage sites; (4) Wash the Anti-eGFP nanobody matrix with a buffer containing the protease corresponding to the restriction site. After the GFP or EGFP tag is removed, the target protein flows down and the purified target protein can be obtained.

6. The method for protein purification using an Anti-eGFP nanobody matrix according to claim 5, characterized in that: The protein purification method also includes regenerating the affinity purification matrix using 6M guanidine hydrochloride or 8M urea.

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