A NFT protein, a preparation method and application thereof, and a purification method of NusA or NusA fusion protein

Abstract

The application provides an NFT protein and a preparation method and application thereof, and a purification method of NusA or NusA fusion protein, and belongs to the field of recombinant protein expression and purification. The NFT protein has a core function of specifically binding to a protein purification tag NusA protein, and can be used as a key tool for targeted binding and efficient purification of NusA fusion protein. Compared with the method of adding a purification tag such as 6xHis in the traditional NusA fusion protein purification method, the NFT protein can be combined with the NusA protein with high affinity and specificity, and can effectively avoid the problem of impurity residue of the purified product caused by non-specific combination, and significantly improve the purity and recovery rate of the NusA fusion protein purification. The NFT protein provided by the application provides a new solution for efficient purification of NusA fusion protein, and has important application value in the fields of recombinant protein drug research and development, industrial enzyme production and life science research.

Description

Technical Field

[0001] This invention belongs to the field of recombinant protein expression and purification, specifically relating to an NFT protein, a recombinant vector, recombinant genetically engineered bacteria and their preparation methods and applications, as well as a purification method for a NusA-containing or NusA-containing fusion protein. Background Technology

[0002] In the field of recombinant protein expression and purification, fusion tag technology has been widely used to improve the solubility, stability, and purification efficiency of target proteins. Among them, the NusA (N-utilization substance A) tag, as a highly efficient E. coli-derived solubilizing tag, can significantly enhance the correct folding and soluble expression of poorly soluble recombinant proteins, especially suitable for the production of eukaryotic or complex structural proteins. However, the NusA tag itself lacks specific affinity ligands and cannot be directly purified by affinity chromatography. In practical applications, it often needs to be combined with small molecule tags such as histidine tags (His-tag) and relies on metal chelate chromatography (such as Ni-NTA resin) for capture. Traditional methods have inherent drawbacks such as non-specific adsorption, risk of metal ion residue, complex processes, and limitations in scalability. To overcome these bottlenecks, researchers have recently attempted to develop various non-metal-dependent purification strategies, such as systems based on enzyme digestion tags, environmentally responsive polymer tags, or antibody affinity tags. However, these methods still suffer from high costs, poor stability, or insufficient versatility. Therefore, there is an urgent need to develop a highly efficient and specific purification method for NusA-tagged proteins that does not require metal chelation chromatography, has a simple process, and is applicable to NusA-tagged proteins. Summary of the Invention

[0003] This invention addresses the current problems by providing an application of NusA-binding protein NFT for purifying NusA prokaryotic expression solubilizing tags and related proteins. This invention aims to solve the problems of metal ion residue, non-specific adsorption, and complex processes caused by the reliance on metal chelate chromatography (such as Ni-NTA) for NusA-tagged protein purification in existing technologies. Traditional methods require the use of His tags and purification via metal columns, resulting in low product safety, insufficient purity, and difficulties in large-scale production.

[0004] This invention proposes a novel purification strategy for NusA-tagged proteins based on the principles of protein-protein interactions. The core of this strategy lies in utilizing the natural interaction between NusA and its specific binding protein, NFT, to construct an MBP-NFT / NusA-tagged protein complex. The complex is then captured in a single step using the affinity of MBP for amylose resin. Finally, high-purity NusA-tagged proteins are obtained through gentle salt gradient elution (e.g., NaCl). Besides the MBP-conjugated NFT protein used in this invention, affinity purification of NFT-NusA fusion proteins can also be achieved by conjugating NFT to agarose matrix or other matrices.

[0005] The specific technical solution is as follows:

[0006] The present invention provides an NFT protein, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] Furthermore, the NFT protein can specifically bind to the NusA protein, enabling targeted binding and purification of the NusA fusion protein.

[0008] Furthermore, when used to purify the NusA fusion protein, it is not necessary to rely on the 6×His or other purification tags on the NusA fusion protein.

[0009] The present invention also provides the use of the above-mentioned NFT protein in recombinant protein drug development, industrial enzyme production or life science research.

[0010] Furthermore, the use of the described NFT protein in the purification of NusA or NusA fusion proteins.

[0011] The present invention also provides a method for preparing NFT protein, the method comprising the following steps: cloning the coding sequence of the NFT protein shown in SEQ ID NO:1 into an expression vector to obtain an NFT recombinant expression vector; introducing the NFT recombinant expression vector into competent host cells to obtain recombinant engineered bacteria; culturing the recombinant engineered bacteria to induce NFT protein expression; centrifuging and purifying to obtain NFT protein.

[0012] The present invention also provides a method for NusA or a purified NusA fusion protein, the method comprising the following steps:

[0013] (1) The above-mentioned NFT protein is combined with a capture reagent to obtain an NFT affinity medium. The NFT affinity medium is combined with a system containing NusA or NusA fusion protein to form a system containing an NFT / NusA complex. The capture reagent is a reagent that can bind to the filler material of affinity chromatography.

[0014] (2) The NFT affinity medium in the system containing the NFT / NusA complex was separated by affinity chromatography to obtain the NusA fusion protein.

[0015] Furthermore, the binding method is selected from chemical coupling or physical interaction; the capturing reagent is selected from maltose-binding protein (MBP), glutathione, N-hydroxysuccinimide (NHS), or agarose matrix; the packing material for affinity chromatography is selected from starch resin (Amylose), glutathione resin (GST Resin), calmodulin resin (Calmodulin Resin), protein A resin (Protein A Resin), or magnetic beads.

[0016] Furthermore, the physical interactions include protein interactions as well as hydrogen bonds, hydrophobic interactions, van der Waals forces, ionic bonds, and other non-covalent interactions such as π-π stacking.

[0017] Furthermore, the affinity chromatography purification includes the following steps: elution with a 30-1000 mM sodium chloride solution as the eluent, and collection of the eluent.

[0018] Furthermore, the concentration of the sodium chloride solution is 30, 50, 100, 300, 500, or 1000 mM.

[0019] Furthermore, in the affinity chromatography purification, before eluting with a 30-1000 mM sodium chloride solution, the following step is also included: rinsing with Buffer A25 containing 5-15 mM benzyl sulfonyl fluoride.

[0020] Furthermore, the concentration of the benzyl sulfonyl fluoride is 10 mM.

[0021] The amino acid sequence of the NFT protein provided by this invention is shown in SEQ ID NO:1:

[0022] The amino acid sequence of SEQ ID NO:1 is as follows:

[0023] DAQTRRRERRAEKQAQWKAANPLLVGVSAKPVNRPILSLNRKPKSRVESALNPIDLTVLAEYHKQIESNLQRIERKGGTQLTPEEKLLRAIFGEK

[0024] The amino acid sequence of the NusA protein provided by this invention is shown in SEQ ID NO:2:

[0025] The amino acid sequence of SEQ ID NO:2 is as follows:

[0026] MNKEILAVVEAVSNEKALPREKIFEALESALATATKKKYEQEIDVRVQIDRKSGDFDTFRRWLVVDEVTQPTKEITLEAARYEDESLNLGDYVEDQIESVTFDRITTQTAKQVIVQKVREAER AMVVDQFREHEGEIITGVVKKVNRDNISLDLGNNAEAVILREDMLPRENFRPGDRVRGVLYSVRPEARGAQLFVTRSKPEMLIELFRIEVPEIGEEVIEIKAAARDPGSRAKIAVKTNDKRIDP VGACVGMRGARVQAVSTELGGERIDIVLWDDNPAQFVINAMAPADVASIVVDEDKHTMDIAVEAGNLAQAIGRNGQNVRLASQLSGWELNVMTVDDLQAKHQAEAHAAIDTFTKYLDIDEDFAT VLVEEGFSTLEELAYVPMKELLEIEGLDEPTVEALRERAKNALATIAQAQEESLGDNKPADDLLNLEGVDRDLAFKLAARGVCTLEDLAEQGIDDLADIEGLTDEKAGALIMAARNICWFGDEA

[0027] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0028] (1) Completely avoid metal ion contamination and ensure the biosafety of the product.

[0029] In the traditional Ni-NTA chromatography process, Ni 2+ Co 2+ Metal ions may detach from the resin and remain in the final product. These metal ions can not only catalyze oxidation reactions, leading to protein structural damage and loss of function, but also pose immunogenic risks in the production of therapeutic proteins (such as vaccines and antibodies). This invention completely avoids the metal chromatography step, using only biocompatible linear starch resin and a NaCl elution buffer system, fundamentally eliminating metal ion contamination and meeting the stringent standards for pharmaceutical-grade protein production.

[0030] (2) Dual specificity ensures high-precision targeted purification

[0031] This invention cleverly combines two highly specific interactions: the high affinity binding of MBP to amylose resin (dissociation constant Kd ≈ 10). -6M); NFT interacts with the native protein of NusA (experimentally verified to be specific). This dual screening mechanism greatly reduces non-specific adsorption of host proteins. Experiments have shown that this system can effectively capture target complexes even in complex bacterial lysate environments.

[0032] (3) The integrated and simplified process significantly reduces operating costs and time.

[0033] Traditional methods require multiple steps, including "His tag purification → enzymatic digestion to remove the tag → secondary purification to remove the tag protein." This invention integrates capture and separation into a single step.

[0034] No protease cleavage required: Avoids the use of expensive TEV / PreScission proteases;

[0035] No secondary purification required: The NFT-NusA complex can be gently separated by optimizing the salt gradient (e.g., NaCl);

[0036] (4) Strong scalability and versatility provide a new paradigm for protein purification.

[0037] This platform technology is not only applicable to NusA-tagged proteins, but can also be extended to other systems by replacing interacting protein pairs:

[0038] Alternative tagging systems: For example, replacing MBP-NFT with GST-NFT allows purification to be performed using glutathione resin. By coupling NFT to NHS or epoxy resin-activated agarose matrix, purification can be achieved without relying on other affinity tags and matrices.

[0039] Reverse purification strategy: Provides a new approach to "reverse purification" for traditional tags such as MBP and GST (i.e., purifying the target protein through interacting proteins, rather than directly binding to the affinity tag).

[0040] Applications for poorly soluble proteins: Combining the solubilizing properties of NusA with the purification advantages of this invention, an integrated "expression-purification" solution is provided for proteins that are difficult to express.

[0041] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0042] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0043] Figure 1 This is the result of low-dose induction of MBP-NFT protein in this invention.

[0044] Figure 2 This is the result of low-dose induction of NusA protein in this invention.

[0045] Figure 3 This is the result of high-dose amylose affinity chromatography purification of MBP-NFT protein in this invention.

[0046] Figure 4 This is the result of high-dose amylose affinity chromatography purification of NusA protein in this invention.

[0047] Figure 5 This is the result of amylose affinity chromatography purification after incubation of NusA protein and NFT in this invention.

[0048] Figure 6 This is the result of amylose affinity chromatography dissociation of NusA protein after incubation with NFT mixture in this invention. Detailed Implementation

[0049] The following detailed description of specific embodiments is provided in conjunction with the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0050] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0051] 1. The strains, reagents, and culture media involved in the examples are as follows:

[0052] The host bacterium used in the experiment was Escherichia coli BL21(DE3), which was purchased from Qingke Biotechnology.

[0053] 2. The main reagents used in the experiment include:

[0054]

[0055] The culture medium used in the experiment was LB (Luria broth) liquid medium (1 L): 10 g peptone, 5 g yeast powder, 10 g NaCl, ddH2O added to 1 L, autoclaved at 121℃ for 20 min.

[0056] Example 1: Construction of the MBP-NFT / NusA tag protein complex

[0057] 1. Transform the prokaryotic expression vector carrying the target gene into the host bacteria.

[0058] Using traditional Ca 2+The heat shock transformation method involved adding plasmids containing the nucleotide sequence encoding the NusA protein and pET43a plasmid containing the nucleotide sequence encoding the MBP-NFT protein (nucleotide sequence shown in SEQ ID NO.3) to BL21(DE3) competent cells, mixing thoroughly, and incubating on ice for at least 30 min. Then, the competent cells were heat-shocked at 42 °C for 90 s in a constant temperature metal bath. Immediately after heat shock, the cells were incubated on ice for 2 min. Then, 800 μL of antibiotic-free LB liquid medium was added, and the cells were cultured on a shaking incubator at 37 °C for 50 min. Afterward, the cells were centrifuged at 12000 g for 1-2 min, discarding part of the supernatant. The cells were resuspended in 100 μL of the supernatant and then spread evenly on solid LB medium containing ampicillin (Amp) (100 μg / ml). The cells were then incubated at 37 °C for 12-18 minutes. h, BL21(DE3) bacterial suspensions carrying NusA and BL21(DE3) bacterial suspensions carrying MBP-NFT were obtained respectively.

[0059] SEQ ID NO.3:

[0060]

[0061] 2. Induction of target protein expression

[0062] (1) Pick BL21(DE3) carrying NusA and BL21(DE3) carrying MBP-NFT respectively, and clone them into 5-10 mL of LB medium containing Amp, and culture at 37℃ until the growth plateau.

[0063] (2) Take 6 ml of each bacterial culture into 600 mL of LB medium containing Amp, and culture in a constant temperature shaking incubator at 37 °C until the OD600 value is 0.5. Then add IPTG to a final concentration of 0.5 mM and incubate overnight at the corresponding temperature (16 °C for MBP-NFT and 37 °C for NusA).

[0064] (3) The next day, centrifuge at 4000 rpm for 10 min to collect the bacterial cells, wash them once with sterile water, centrifuge under the same conditions, and retain the bacterial cells;

[0065] (4) Add 30 mL of Buffer A25 and 1 / 100 volume of 0.1 M PMSF to resuspend the bacterial cells, and use the No. 6 amplitude bar of the ultrasonic cell disruptor to sonicate at 60% power for 30 min.

[0066] (5) After centrifuging at 12,000 rpm for 30 min at 4℃ using a high-speed refrigerated centrifuge, the supernatant was collected and stored at 4℃ to obtain bacterial supernatant containing the target protein NusA (theoretical molecular weight 57 KDa, apparent molecular weight 70 KDa) and MBP-NFT (54 KDa).

[0067] 3. Constructing the MBP-NFT / NusA tag protein complex

[0068] The target protein NusA and MBP-NFT bacterial supernatant were mixed at a volume ratio of 1:4 and incubated at 4°C for 4 hours to obtain bacterial supernatant containing the MBP-NFT / NusA tag protein complex.

[0069] Example 2: Purification of NusA tag protein using the MBP-NFT / NusA tag protein complex

[0070] I. Experimental Principle

[0071] The MBP-NFT fusion protein binds to amylose resin with high affinity via its MBP tag, forming a stable "resin-MBP-NFT / NusA" complex. Under high ionic strength NaCl conditions, the non-covalent interaction between NFT and NusA is weakened, while MBP and amylose remain bound. This allows for the selective release of NusA into the permeation solution, achieving one-step capture and simultaneous acquisition of high-purity NusA-tagged protein.

[0072] II. Solution Preparation

[0073] Eluent: NaCl solutions of different concentrations (30, 50, 100, 300, 500, 1000 mM) (the NaCl solutions also contain 20 mM Tris-HCl, pH=8.0), with an elution volume of 5 column volumes for each concentration of NaCl solution. Column volume refers to the total volume of the packing medium in the chromatography column; those skilled in the art can select the appropriate column size according to the required scale, while maintaining an elution volume of 5 column volumes.

[0074] III. Operating Procedures

[0075] A method for purifying NusA-tagged protein includes: (1) loading the bacterial supernatant containing the MBP-NFT / NusA-tagged protein complex into amylose resin; (2) washing the amylose resin with 20 mL Buffer A25 (containing 10 mM PMSF); and (3) washing the amylose resin with 30-1000 mM NaCl and collecting the effluent.

[0076] Figure 6 The results showed that the NusA protein could be obtained by gradient elution with NaCl (30-1000 mM), while the MBP-NFT remained in the packing material.

[0077] The following experimental examples demonstrate the beneficial effects of the present invention.

[0078] Experiment 1: Expression of the target protein and determination of the induction temperature

[0079] 1. Expression of the target protein and determination of the induction temperature

[0080] (1) Pick BL21(DE3) carrying NusA and BL21(DE3) carrying MBP-NFT respectively, and clone them into 5-10 mL of LB medium containing Amp, and culture at 37℃ until the growth plateau.

[0081] (2) Take 100 μL of each of the two bacterial cultures expressing NusA and MBP-NFT proteins and add them to 10 mL of LB medium containing Amp. Incubate at 37°C in a shaking incubator until the OD600 value is 0.5 and then divide them into groups (the grouping is shown in Table 1).

[0082] (3) IPTG was added to the experimental groups of the two bacterial cultures to a final concentration of 0.5 mM, while no IPTG was added to the control group. The bacterial cultures of MBP-NFT protein were cultured overnight in constant temperature shakers at 16℃ and 37℃, respectively, and the bacterial cultures expressing NusA protein were cultured overnight in constant temperature shakers at 37℃.

[0083] (4) The next day, centrifuge at 4000 rpm for 10 min to collect the bacterial cells, wash them once with sterile water, centrifuge under the same conditions, and retain the bacterial cells;

[0084] (5) Add 1 mL of Buffer A25 and 1 / 100 volume of 0.1 M PMSF to resuspend the bacterial cells, and use the No. 3 amplitude bar of the ultrasonic cell disruptor to sonicate at 30% power for 10 min.

[0085] (6) Centrifuge at 4℃ and 12000 rpm for 30 min using a benchtop refrigerated centrifuge, then collect the supernatant and store it at 4℃.

[0086] 2. SDS-PAGE and Result Detection

[0087] (1) Mix the prepared 2× protein loading buffer with the preserved protein supernatant thoroughly and boil for 10 min;

[0088] (2) Prepare 10% separating adhesive, leaving 2-3 cm between the adhesive surface and the top surface of the glass plate, and add isopropanol for pressing;

[0089] (3) After the separating gel solidifies, the isopropanol is dried with filter paper, then the prepared 5% concentrated gel is added, and the mold comb is inserted. It can be used after the gel solidifies.

[0090] (4) Assemble the electrophoresis tank, pour in the electrophoresis buffer, pull out the mold comb vertically upwards, add non-prestained protein marker (2 μL / well) to one well first, and then add appropriate concentrations of protein sample (10 μL / well) to the remaining wells in sequence.

[0091] (5) After loading the sample, close the electrophoresis apparatus lid, maintain a constant voltage of 80 V for 30 min, then change the voltage to 120 V for 60 min to complete the electrophoresis.

[0092] (6) The PAGE gel after electrophoresis was stained with Coomassie Brilliant Blue at 37°C for 1 h;

[0093] (7) After staining, discard the staining solution, pour in the decolorizing solution, and decolorize at 37°C for 1 h;

[0094] (8) After destaining, use a gel imaging system to expose and test whether NusA and MBP-NFT proteins are successfully expressed.

[0095] Table 1

[0096]

[0097] according to Figure 1 , Figure 2 The experimental results show that MBP-NFT can induce the target protein (MBP-NFT, 54 KDa) at 16℃ + 0.5 μM IPTG, and NusA can induce the target protein (NusA, theoretical molecular weight 57 KDa, apparent molecular weight 70 KDa) at 37℃ + 0.5 μM IPTG.

[0098] Experiment Example 2: Verification using NusA and MBP

[0099] (1) Pick BL21(DE3) carrying NusA and BL21(DE3) carrying MBP-NFT respectively, and clone them into 10 mL of LB medium containing Amp. Incubate at 37℃ until the growth plateau.

[0100] (2) Take 6 ml of each bacterial culture into 600 mL of LB medium containing Amp, and culture in a constant temperature shaking incubator at 37 °C until the OD600 value is 0.5. Then add IPTG to a final concentration of 0.5 mM and incubate overnight at the corresponding temperature (16 °C for MBP-NFT and 37 °C for NusA).

[0101] (3) The next day, centrifuge at 4000 rpm for 10 min to collect the bacterial cells, wash them once with sterile water, centrifuge under the same conditions, and retain the bacterial cells;

[0102] (4) Add 30 mL of Buffer A25 and 1 / 100 volume of 0.1 M PMSF to resuspend the bacterial cells, and use the No. 6 amplitude bar of the ultrasonic cell disruptor to sonicate at 60% power for 30 min.

[0103] (5) Centrifuge at 12,000 rpm for 30 min using a high-speed refrigerated centrifuge at 4℃, then collect the supernatant and store it at 4℃;

[0104] (6) Take half of the obtained NusA and MBP-NFT and perform amylose affinity column chromatography separately; take each component sample for SDS-PAGE verification (the results are as follows). Figure 3 , Figure 4(as shown)

[0105] (7) The other half of the supernatant of the two proteins were mixed at a volume ratio of NusA:MBP-NFT=1:4 and incubated at 4℃ for 4h. Then, amylose affinity chromatography was performed, and each fraction sample was verified by SDS-PAGE (the results are shown in the figure). Figure 5 (As shown).

[0106] The experimental results showed that the proteins expressed by the two strains carrying the expression vectors did not appear at the protein marker 70 kDa in the elution fraction obtained by amylose affinity chromatography. NusA protein (the protein at the protein marker 70 kDa mentioned above) only appeared in the elution fraction after the two strains were incubated.

[0107] In summary, this invention provides an NFT protein, its preparation method, and its applications, as well as a purification method for NusA or NusA fusion proteins. The NFT protein of this invention possesses the core function of specifically binding to the NusA protein purification tag, serving as a key tool for the targeted binding and efficient purification of NusA fusion proteins. Compared to traditional NusA fusion protein purification methods that rely on adding additional purification tags such as 6×His, the NFT protein can bind to the NusA protein with high affinity and specificity, effectively avoiding the problem of impurities in the purification product caused by non-specific binding, and significantly improving the purity and recovery rate of NusA fusion protein purification. The NFT protein provided by this invention offers a novel solution for the efficient purification of NusA fusion proteins, and has significant application value in recombinant protein drug development, industrial enzyme production, and life science research.

[0108] Although the present invention has been shown and described through specific embodiments, it should be understood that the above embodiments are for illustration only and not for limiting the invention. Those skilled in the art can make various modifications, adjustments, and variations in form and detail without departing from the spirit and scope of the invention as defined by the appended claims and their equivalents. Any simple variations, equivalent substitutions, or modifications based on the essence of the present invention are covered within the protection scope of the present invention.

Claims

1. An NFT affinity medium, characterized in that, The NFT affinity medium is obtained by fusing MBP protein, Linker and NFT protein; wherein, MBP protein is located at the N-terminus of the NFT affinity medium, NFT protein is located at the C-terminus of the NFT affinity medium, and the amino acid sequence of NFT protein is shown in SEQ ID NO:

1.

2. The use of the NFT affinity medium according to claim 1 in the purification of NusA protein.

3. A method for preparing NFT affinity media, characterized in that, The method includes the following steps: preparing an MBP-NFT recombinant expression vector with a nucleotide sequence as shown in SEQ ID NO:3; introducing the MBP-NFT recombinant expression vector into competent host cells to obtain recombinant engineered bacteria; culturing the recombinant engineered bacteria to induce NFT affinity mediator expression; centrifuging and purifying to obtain NFT affinity mediator.

4. A method for purifying NusA protein, characterized in that, The method includes the following steps: (1) The NFT affinity medium of claim 1 is combined with a system containing NusA protein to form a system containing MBP-NFT / NusA complex; (2) The system containing the MBP-NFT / NusA complex was loaded onto starch resin, rinsed, eluted, and the effluent was collected to obtain NusA protein.

5. The method according to claim 4, characterized in that, The starch resin is a linear starch resin.

6. The method according to claim 4, characterized in that, The elution was performed using a 30-1000 mM sodium chloride solution as the eluent.

7. The method according to claim 6, characterized in that, The concentrations of the sodium chloride solutions are 30, 50, 100, 300, 500, and 1000 mM.

8. The method according to claim 4, characterized in that, The rinsing is performed using Buffer A25 containing 5-15 mM benzyl sulfonyl fluoride.

9. The method according to claim 8, characterized in that, The concentration of the benzyl sulfonyl fluoride is 10 mM.

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