Bifunctional protein tag for improving thermal stability and solubility of recombinant protein, product based on bifunctional protein tag and application of bifunctional protein tag

Abstract

The invention discloses a bifunctional protein tag for improving thermal stability and solubility of recombinant protein, a product based on the bifunctional protein tag and application of the bifunctional protein tag, and belongs to the technical field of protein engineering. The amino acid sequence of the bifunctional tag is shown as SEQ ID NO: 1, the bifunctional tag has the similarity of 70%-95% or above, and the amino acid sequence variation mainly exists but is not limited to a secondary structure position. The bifunctional tag can be used at an N terminal or a C terminal of a target protein, and is suitable for expression and purification of a fusion protein in a prokaryotic expression system. The hydrophobic region of the natural protein is transformed through rational design, so that the technical problems that the existing protein tag is large in molecular weight and easy to shield the active structural domain of the target protein, and the residual non-natural amino acid after the tag is removed possibly destroys the correct folding of the protein are solved; the solubility and the thermal stability of the fusion protein in an escherichia coli expression system are remarkably improved, and the method is suitable for functional expression and industrial application of inclusion body protein.

Description

Technical Field

[0001] The present invention belongs to the field of protein engineering technology, and particularly relates to a bifunctional protein tag for improving the thermal stability and solubility of a recombinant protein, and products and applications based thereon. Background Art

[0002] Recombinant proteins are widely used in fields such as biomedicine, industrial enzyme preparation, and scientific research. However, they often face problems such as low solubility and poor thermal stability during expression, which seriously limit their industrial application. To address these problems, researchers have developed a variety of protein tagging technologies. By fusing specific amino acid sequences to the N-terminus or C-terminus of the target protein, the solubility expression and stability of the recombinant protein are improved. Therefore, protein tagging technology is a key tool for recombinant protein expression. By fusing specific amino acid sequences to the N-terminus or C-terminus of the target protein, it can achieve functions such as affinity purification, activity detection, and stability enhancement. Currently, commonly used protein tags include maltose binding protein (MBP), glutathione S-transferase (GST), and NusA. These tags can improve the solubility expression of the target protein to a certain extent. For example, Chinese invention patent publication number CN116589591A discloses the use of MBP tags, Spy tags, or MBP-Spy tandem tags in assisting recombinant protein expression. The solubility-promoting effect of the MBP-Spy tandem tag is significantly better than that of the Spy tag or MBP tag alone. The Chinese invention patent with publication number CN118599792A describes a new type of solubility-promoting tag, which is inserted into a prokaryotic expression vector together with the TEV protease recognition site, which can significantly promote the soluble expression of the target protein. In addition, the Chinese invention patent with publication number CN116444622A discloses a polypeptide composed of 4-6 specific amino acids, which has at least 20% α-helical structure, which can significantly improve the soluble expression level of the fusion protein and prolong the half-life of the drug protein. The Chinese invention patent with publication number CN114381471A proposes the use of an auxiliary protein in the production of recombinant proteins. The system has high expression efficiency, can express recombinant proteins soluble in cells, will not form inclusion bodies, and can achieve cell disruption by heating the bacteria. The patent with international patent number WO2024119434A1 describes an acidic surface solubility-promoting short peptide tag that can improve the expression efficiency of recombinant proteins.

[0003] In recent years, the development of small molecular weight solubility-enhancing tags has become a breakthrough. For example, the 20 kDa domain of the translation initiation factor IF2 has been shown to maintain target protein activity, but its solubility-enhancing effect remains unstable. Current protein tags still have many limitations: First, most solubility-enhancing tags have a large molecular weight (e.g., MBP approximately 42 kDa, GST approximately 26 kDa, and NusA approximately 55 kDa), which can easily mask the active domain of the target protein, significantly reducing enzymatic activity. Second, the unnatural amino acids remaining after tag removal may interfere with or disrupt proper protein folding. Third, existing tags primarily focus on improving protein solubility, with limited improvement in thermal stability, especially for large molecular weight proteins above 15 kDa. Fourth, there is a disconnect between existing solubility-enhancing mechanisms and actual effectiveness. While some fusion proteins can improve protein solubility, the improvement in thermal stability under harsh conditions such as high temperature, extreme pH, or organic solvents is limited, making them difficult to meet industrial requirements.

[0004] Therefore, developing a new fusion tag with a small molecular weight, high solubility-promoting efficiency and simultaneous enhanced thermal stability is of great significance to promoting the large-scale application of recombinant proteins. Summary of the Invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a bifunctional protein tag that improves the thermal stability and solubility of recombinant proteins, as well as products and applications based thereon, so as to solve the technical problem that protein tags in the prior art cannot simultaneously meet the requirements of thermal stability and solubility.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention discloses a bifunctional protein tag for improving the thermal stability and solubility of a recombinant protein, which is a protein as described below: a) a protein with the amino acid sequence shown in SEQ ID NO: 1; b) a protein having an amino acid sequence that is greater than 70% to 90% identical to SEQ ID NO: 1 after substitution and / or deletion and / or addition of amino acid residues to the sequence shown in SEQ ID NO: 1 and having the same function as a); c) A protein formed by splicing of a) and / or b) and having the same function as a).

[0007] Preferably, the bifunctional protein tag is assembled into a stable trimer structure by multiple α-helices and β-pleated sheets according to the topological arrangement of β1-α1-β2-β3-η1-α2-β4-α3; wherein the β-pleated sheets are arranged in a cis-parallel manner and stabilized by a hydrogen bond network, and the α-helices form stable helical bundles through side chain hydrophobic interactions and interchain hydrogen bonds.

[0008] Preferably, the amino acid sequence variation mainly exists but is not limited to the secondary structure position.

[0009] Further preferably, the sequence mutation mode with more than 70%-90% identity with SEQ ID NO: 1 includes: the 27th amino acid sequence is I / A, the 30th amino acid sequence is A / F / V, the 33rd amino acid sequence is R / F / V / I, the 38th amino acid sequence is G / F / I / L, the 40th amino acid sequence is W / R / Y, the 44th amino acid sequence is E / K, the 45th amino acid sequence is I / V / L, the 46th amino acid sequence is E / V, the 52nd amino acid sequence is G / A / Q, the 60th amino acid sequence is D / E / S, the 61st amino acid sequence is L / K / A / H, the 62nd amino acid sequence is W / E / Q, the 64th amino acid sequence is E / A, the 66th amino acid sequence is K / L, the 68th amino acid sequence is R / E / C, the 69th amino acid sequence is I / L / V, the 70th amino acid sequence is K / R, the 71st amino acid sequence is E / K / S, the 72nd amino acid sequence is L / H, the 76th amino acid sequence is D / E / Q, the 77th amino acid sequence is V / T, the 79th amino acid sequence is A / E / C, the 80th amino acid sequence is I / L, the 81st amino acid sequence is I / F / V / L, the 82nd amino acid sequence is R / T / A / V, the 83rd amino acid sequence is I / L, the 84th amino acid sequence is D / P, the 85th amino acid sequence is V / I, the 86th amino acid sequence is D / E / T / A, the 87th amino acid sequence is D / N / E / H / Q, the 88th amino acid sequence is V / G, the 89th amino acid sequence is N / L / D, the 94th amino acid sequence is K / N / D / S, and the 97th amino acid sequence is I / N / R.

[0010] The application also discloses application of the above-mentioned bifunctional protein tag for improving thermal stability and solubility of a recombinant protein in protein soluble expression.

[0011] The application also discloses a fusion protein comprising at least a target protein and the above-mentioned bifunctional protein tag for improving thermal stability and solubility of a recombinant protein, and the bifunctional protein tag for improving thermal stability and solubility of a recombinant protein is at the N terminal or C terminal of the target protein.

[0012] Preferably, a protease recognition site and / or a purification tag are further comprised between the target protein and the bifunctional protein tag for improving thermal stability and solubility of a recombinant protein.

[0013] The application also discloses an expression vector, which is a prokaryotic expression vector containing the above-mentioned fusion protein or a coding gene thereof.

[0014] Preferably, the prokaryotic expression vector can be a prokaryotic expression vector such as a pET series vector, a pGEX series vector, a pMAL series vector, or a pCold series vector.

[0015] The present invention also discloses a host cell comprising the above expression vector, and the host cell can express the fusion protein.

[0016] The present invention also discloses the application of the above fusion protein, expression vector or host cell in protein expression and purification.

[0017] Compared with the prior art, the present invention has the following beneficial effects: By engineering the hydrophilicity of the natural heat-resistant protein CutA, this invention designs a bifunctional protein tag, nCUTA, that can synergistically improve the solubility and thermal stability of fusion proteins. This provides an innovative solution for the functional expression of inclusion body proteins. The advantages of this innovative invention are mainly reflected in: 1) Significantly Improved Inclusion Body Protein Solubility: By sequence-modifying the thermostable CutA protein, the resulting nCUTA protein tag significantly enhances the soluble expression of inclusion body proteins. Experimental data showed that when the pCold–nCUTA-FT fusion was expressed in the Rosetta expression strain, the proportion of soluble protein in the supernatant was 1.82 times that of wild-type pCold-FT, comparable to the solubility-enhancing effect of the NusA tag, thus resolving the problem of poor inclusion body protein solubility in existing technologies. 2) Significantly improve the thermal stability of the fusion protein: The bifunctional protein tag of the present invention can significantly improve the storage conditions and thermal aggregation resistance of the fusion protein. At the same time, it allows the fusion protein to react at a higher temperature, improving the reaction efficiency and solving the problem of low thermal stability of fusion proteins in the prior art. 3) Small molecular weight, less impact on fusion protein activity: Compared with the large molecular weight protein tags in the prior art, the nCUTA tag of the present invention has a smaller molecular weight. Without removing the protein tag, it can still maintain high biological activity. However, large molecular weight tags such as NusA have a greater impact on the activity of fusion proteins. This solves the problem of large molecular weight protein tags in the prior art that have a significant impact on the activity of fusion proteins. 4) Wide range of applications: Compared with traditional protein tags that can only improve the thermal stability of proteins within 15Kd, the bifunctional protein tag provided by the present invention can improve the thermal stability of proteins above 100Kd, greatly expanding the scope of application and solving the problem of limited application range of traditional protein tags in the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a multiple sequence comparison diagram of CUTA-related proteins; Figure 2Comparison of CUTA monomer structure predictions. In Figure A, the left side shows the CUTA monomer, the middle side shows the nCUTA monomer, and the right side shows the alignment result. In Figure B, the left side shows the PHCUTA monomer, the middle side shows the nCUTA monomer, and the right side shows the alignment result. Figure 3 Analysis of the hydrophilicity of CUTA and nCUTA; A is the hydrophilicity score of CUTA; B is the hydrophilicity score of nCUTA; Figure 4 The purification results of T4 DNA polymerase and CUTA-T4 DNA polymerase are shown. A shows the purified T4 DNA polymerase protein, which is approximately 107.2 kDa. Lane M is the 26616 protein marker, with lane 1 being before induction, lane 2 being after induction, lane 3 being before column flow, lane 4 being after column flow, and lanes 5-13 being purified protein samples. B shows the purified CUTA-T4 DNA polymerase protein, which is approximately 120.3 kDa. Lane M is the 26616 marker, with lane 1 being before induction, lane 2 being after induction, lane 3 being before column flow, lane 4 being after column flow, and lanes 5-13 being purified protein samples. Figure 5 Figure 2 is the activity detection diagram of T4 DNA polymerase and CUTA-T4 DNA polymerase; A is the non-deformation gel electrophoresis diagram of T4 DNA polymerase and CUTAT4 DNA polymerase after being treated at different temperatures, lane M is a 500 bp marker, lane 1 is commercial T4 DNA polymerase as a positive control; lane 2 is a negative control without adding enzyme; the remaining lanes except the marker are grouped into two groups, the left one is T4 DNA polymerase and the right one is CUTA-T4 DNA polymerase; lanes 3-4 are 53.5℃ group; lanes 5-6 are 53.9℃ group; lanes 7-8 are 55℃ group; lanes 9-10 are 56.3℃ group; lanes 11-12 are 57.3℃ group; lanes 13-14 are 58.5℃ group; lanes 15-16 are 59.6℃ group; lanes 17-18 are 60℃ group; B is the activity detection diagram of T4 DNA polymerase and CUTA-T4 DNA polymerase relative activity statistics; Figure 6 Figure 5 shows the results of FT solubility analysis. Lanes A show the FT group (pre-induction, post-induction, lysis supernatant, and lysis pellet, respectively); lanes 5-8 show the nCUTA-FT group (pre-induction, post-induction, lysis supernatant, and lysis pellet, respectively); lanes 9-12 show the NusA-FT group (pre-induction, post-induction, lysis supernatant, and lysis pellet, respectively); lane M shows the 26616 protein marker. B shows the results of Western blotting (WB) protein analysis, with the same lane layout as in A. C shows the relative expression analysis; and D shows the relative expression analysis.

[0019] Figure 7This is the result of FTase activity detection by high performance liquid chromatography; Figure 8 For FT activity detection. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0021] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that the numbers used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0022] The present invention is described in further detail below with reference to the accompanying drawings: Example 1 This embodiment provides a bifunctional protein tag that can improve the thermal stability and solubility of recombinant proteins. The bifunctional protein tag is composed of multiple α-helices and β-parallel structures β1-α1-β2-β3-η1-α2-β4-α3, and the formed tertiary structure can form a stable trimer structure.

[0023] The amino acid sequence of the bifunctional protein tag has a similarity of 70% to 95% or more to the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence shown in SEQ ID NO: 1 is: EIVVYTTVPDEESAQDLAKTLLEEKLAACVNLREHTSFYWWEGKLEQDKEVQLMILKTTSHQQELKEELKSHHPYQTPEILRLPVDHGNEDYLKWLREETRK.

[0024] CutA divalent-cation tolerance protein (CUTA), a bifunctional protein tag capable of improving the thermal stability and solubility of recombinant proteins, is derived from various species, including but not limited to Archaeoglobus fulgidus, Ehrlichia chaffeensis, Escherichia coli, Homosapiens, Oryza sativa subsp. Japonica, Pyrococcus horikoshii, Rattus norvegicus, Salmonella typhimurium, Thermotoga maritima, Thermus thermophilus, and Yersiniapestis. Through multiple sequence alignment, the highly hydrophilic nCUTA protein, which maintains its original structure, was identified and named nCUTA (new CUTA). This bifunctional protein tag, designed to enhance the thermal stability and solubility of fusion proteins, was named nCUTA (new CUTA).

[0025] The amino acid sequence variation of the bifunctional protein tag mainly exists but is not limited to the secondary structure position. Specifically, the amino acid sequence mutation mode of the bifunctional protein tag mainly includes: the 27th amino acid sequence can be I / A, the 30th amino acid sequence can be A / F / V, the 33rd amino acid sequence can be R / F / V / I, the 38th amino acid sequence can be G / F / I / L, the 40th amino acid sequence can be W / R / Y, the 44th amino acid sequence can be E / K, the 45th amino acid sequence can be I / V / L, the 46th amino acid sequence can be E / V, the 52nd amino acid sequence can be G / A / Q, the 60th amino acid sequence can be D / E / S, the 61st amino acid sequence can be L / K / A / H, the 62nd amino acid sequence can be W / E / Q, the 64th amino acid sequence can be E / A, the 66th amino acid sequence can be K / L, the 68th amino acid sequence can be R / E / C, the 69th amino acid sequence can be I / L / V, the 70th amino acid sequence can be The amino acid sequence may be K / R, the 71st amino acid sequence may be E / K / S, the 72nd amino acid sequence may be L / H, the 76th amino acid sequence may be D / E / Q, the 77th amino acid sequence may be V / T, the 79th amino acid sequence may be A / E / C, the 80th amino acid sequence may be I / L, the 81st amino acid sequence may be I / F / V / L, the 82nd amino acid sequence may be R / T / A / V, the 83rd amino acid sequence may be I / L, the 84th amino acid sequence may be D / P, the 85th amino acid sequence may be V / I, the 86th amino acid sequence may be D / E / T / A, the 87th amino acid sequence may be D / N / E / H / Q, the 88th amino acid sequence may be V / G, the 89th amino acid sequence may be N / L / D, the 94th amino acid sequence may be K / N / D / S, and the 97th amino acid sequence may be I / N / R.

[0026] When used for prokaryotic expression and purification of fusion proteins, this bifunctional protein tag comprises a purification tag, a bifunctional tag, and a target protein. The bifunctional tag can be located at either the N-terminus or the C-terminus of the target protein. Prokaryotic expression vectors can include pET, pGEX, pMAL, and pCold vectors.

[0027] 1. Structural characteristics of bifunctional protein tags The bifunctional protein tag in this embodiment is designed and modified from CutA protein, which is a small molecular weight protein widely existing in various organisms and has extremely high thermal stability. Through structural analysis of CutA protein, it is found that it is composed of β1-α1-β2-β3-η1-α2-β4-α3 multiple secondary structure units, which form a stable trimer structure through specific spatial arrangement.

[0028] The tertiary structure characteristics of the bifunctional protein tag mainly manifest as follows: three identical monomers form a stable trimer through non-covalent interaction, and each monomer is composed of two α helices and four β folds. The β fold regions of the three monomers interact to form a tight hydrophobic core, while the α helices are exposed on the outer surface of the trimer. This special structural arrangement makes the protein tag have extremely high thermal stability, and it can still maintain its structural integrity even at high temperature of 100℃.

[0029] Through analysis of the original CutA protein sequence, this embodiment is systematically modified, mainly focusing on the following aspects: 1) Enhancing surface hydrophilicity: by replacing the surface-exposed hydrophobic amino acids with hydrophilic amino acids, the solubility of the protein tag is improved; 2) Optimizing core hydrophobic interaction: by precisely adjusting the hydrophobic amino acids in the core region, the stability of the trimer structure is enhanced; 3) Introducing favorable electrostatic interaction: by introducing charged amino acids at appropriate positions, additional salt bridges are formed, further stabilizing the protein structure.

[0030] These modifications make the bifunctional protein tag of this embodiment maintain high thermal stability while significantly improving its solubility, thereby effectively improving the expression level and stability of the fusion protein.

[0031] 2. Amino acid sequence characteristics of the bifunctional protein tag The amino acid sequence of the bifunctional protein tag of this embodiment is shown in SEQ ID NO: 1. Through analysis of SEQ ID NO: 1, the positions of each secondary structure unit can be determined, including: - β1 structure: about the 1-8th amino acid; - α1 structure: about the 12-24th amino acid; - β2 structure: about the 28-35th amino acid; - β3 structure: about the 40-47th amino acid; - η1 structure: about the 52-58th amino acid; - α2 structure: about the 62-75th amino acid; - β4 structure: approximately located at amino acids 80-87; - α3 structure: approximately located at amino acids 90-100; The spatial arrangement of these secondary structural units forms a stable trimer structure, which is the structural basis for the function of the bifunctional protein tag.

[0032] 3. Amino acid sequence variation of bifunctional protein tags The bifunctional protein tag in this example allows for amino acid mutations at specific positions, primarily at sites of secondary structural change, i.e., at the junctions between secondary structural units. While amino acid mutations in these regions generally do not significantly affect the overall folding and function of the protein, they can be used to optimize protein performance by introducing specific amino acids.

[0033] Specifically, the bifunctional protein tag of this embodiment allows amino acid substitutions at positions 27, 30, 33, 38, 40, 44, 45, 46, 52, 60, 61, 62, 64, 66, 68, 69, 70, 71, 72, 76, 77, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 94, and 97. Amino acid substitutions at these positions follow the following principles: 1) Conservative substitutions: The substituted amino acid has similar physical and chemical properties to the original amino acid, such as substitutions between hydrophobic amino acids (such as I / L / V) or substitutions between charged amino acids (such as D / E or K / R); 2) Functional optimization substitution: Optimizing the solubility, stability, or other functional properties of a protein by introducing specific amino acids, such as replacing surface-exposed hydrophobic amino acids with hydrophilic amino acids; 3) Structural stabilization substitutions: Enhance the structural stability of the protein by introducing amino acids that can form additional stabilizing interactions, such as hydrogen bonds or salt bridges.

[0034] The combination of these amino acid substitutions can produce multiple variants, each of which maintains the basic structure and function of the bifunctional protein tag but may exhibit different performance advantages in specific application scenarios.

[0035] 4. Application of bifunctional protein tags in fusion protein expression The bifunctional protein tag of this embodiment can be fused with the target protein to form a fusion protein. The design of a fusion protein generally includes three main parts: a purification tag, a bifunctional tag, and the target protein.

[0036] A purification tag is typically located at the N-terminus of the fusion protein for subsequent affinity purification. Common purification tags include the His tag (6-10 histidine residues), GST tag (glutathione S-transferase), and MBP tag (maltose binding protein). In this example, the His tag was preferred as the purification tag because it has a small molecular weight, does not significantly increase the total molecular weight of the fusion protein, and simplifies purification.

[0037] The bifunctional tag can be located at either the N-terminus or the C-terminus of the target protein. When located at the N-terminus, the bifunctional tag can play a role in the early stages of protein translation, guiding the target protein to fold correctly. When located at the C-terminus, the bifunctional tag can stabilize the folded target protein structure. In most applications, placing the bifunctional tag at the N-terminus generally yields better results.

[0038] The target protein is the target protein in the fusion protein that needs to be expressed and purified. The bifunctional protein tag of this embodiment is applicable to various types of target proteins, especially those that easily form inclusion bodies or have low expression levels in conventional expression systems.

[0039] 5. Selection of expression vectors for bifunctional protein tags The bifunctional protein tag of this embodiment can be expressed using a variety of prokaryotic expression vectors, including pET series vectors, pGEX series vectors, pMAL series vectors, and pCold series vectors.

[0040] The pET series of vectors are among the most commonly used prokaryotic expression vectors. They feature a T7 promoter and can efficiently express target proteins under IPTG induction. These vectors are typically used with E. coli expression hosts such as BL21(DE3) to achieve high levels of protein expression.

[0041] The pGEX series vectors contain a GST tag that can be used in combination with a bifunctional tag to form a dual-tag system. The GST tag can not only be used for affinity purification, but can also improve the solubility of fusion proteins in some cases.

[0042] The pMAL series vectors contain the MBP tag, which is a commonly used solubility-promoting tag that can work synergistically with the bifunctional tag to further improve the solubility of the fusion protein.

[0043] The pCold series of vectors are designed for low-temperature expression. They can express proteins at low temperatures (e.g., 15°C), helping to reduce the formation of inclusion bodies. Combining bifunctional tags with pCold vectors can further improve the soluble expression of poorly soluble proteins.

[0044] In this embodiment, the pET series vectors, particularly the pET-28a vector, are preferably used because they have a dual promoter system, can achieve efficient protein expression, and contain His tag sequences, facilitating subsequent affinity purification.

[0045] 6. Expression and purification of bifunctional protein tag In this embodiment, the expression and purification of the bifunctional protein tag fusion protein are as follows: 1) The gene sequence of the purification tag-nCUTA bifunctional protein tag-fusion protein, or the purification tag-fusion protein-nCUTA bifunctional protein tag, or the nCUTA bifunctional protein tag-fusion protein-purification tag, or the fusion protein-nCUTA bifunctional protein tag-purification tag is inserted into a pET series vector, a pGEX series vector, a pMAL series vector, and a pCold series vector, etc. prokaryotic expression vector by double enzyme digestion or homologous recombination.

[0046] 2) The recombinant expression vector is transformed into DH5α competent cells, and after colony PCR identification, positive clones are selected and plasmid DNA is extracted.

[0047] 3) The recombinant plasmid DNA obtained in step 2) is transformed into BL21(DE3), Rosetta or OrigamiB(DE3) expression bacteria by heat shock method. Positive clones are selected and after 37℃ 220rpm overnight culture, 600ml LB medium is added and the culture is continued at 37℃ until OD600≈0.6. Then IPTG is added to a final concentration of 0.5mM and the culture is transferred to 16℃ for overnight culture.

[0048] 4) The overnight culture is centrifuged at 8000 rpm for 5 min to collect the bacterial cells. The resulting bacterial cells are resuspended with Buffer A at a wet weight of 30ml / g, pulsed at 200W for 5 seconds with an interval of 10 seconds for a total of 60 minutes. Then centrifuge at 12000 rpm for 30 min. Take the supernatant, filter through a 0.22μm filter membrane, and then purify by nickel ion affinity chromatography column.

[0049] The buffers used are as follows: Buffer A: 20mM Tris-HCl (pH8.0), 300mM NaCl, 20mM imidazole. Buffer B: 20mM Tris-HCl (pH8.0), 300mM NaCl, 500mM imidazole.

[0050] 5) The specific steps for affinity chromatography are as follows: Inspection and cleaning of the purification instrument: Turn on the AKTA PURE protein purifier and check the waste liquid tank, pump head, water bottle, and automatic sample collector. Set the system pressure and pipeline pressure to 0.5 MPa. Use ultrapure water to flush the system. Click A Wash, B Wash, and System Wash in sequence to flush the pump and pipeline. Set the column direction to downward, close the restrictor, and set the flow rate to 1 ml / min and 50% Buffer B. At this time, the changes in conductivity and UV absorbance can be observed until the conductivity reaches 0 and the UV absorbance stabilizes. Prepare for sample loading. When the UV value rises rapidly, collect the flow-through sample (after passing through the column). After the UV value decreases and stabilizes again, pull the gradient (Buffer B content slowly increases to 100% within 10 minutes). Start collecting the purified protein sample after the UV value rises rapidly again. Stop collecting samples when the UV value decreases and stabilizes again.

[0051] Example 2 This example provides a variant of a bifunctional protein tag, whose amino acid sequence is 85% similar to that of SEQ ID NO: 1. While maintaining the basic structure and function of the original bifunctional protein tag, this variant further optimizes the tag's performance through amino acid substitutions at specific sites.

[0052] Specifically, the variant undergoes amino acid substitutions at the following sites: position 27 is replaced by I to A, position 30 is replaced by A to F, position 33 is replaced by R to V, position 38 is replaced by G to I, position 40 is replaced by W to Y, position 44 is replaced by E to K, position 45 is replaced by I to L, position 46 is replaced by E to V, position 52 is replaced by G to A, position 60 is replaced by D to E, position 61 is replaced by L to K, position 62 is replaced by W to E, position 64 is replaced by E to A, position 66 is replaced by K to L, position 68 is replaced by R to C, position 69 is replaced by I to L, position 70 is replaced by K to R, position 71 is replaced by E to K, and position 72 is replaced by L to H.

[0053] These amino acid substitutions were primarily concentrated at sites with secondary structural changes. By optimizing the amino acid composition at these sites, the solubility and thermal stability of the bifunctional protein tag were further improved. In particular, increasing the proportion of surface hydrophilic amino acids significantly improved the tag's solubility, while optimizing the hydrophobic interactions in the core region enhanced the tag's thermal stability.

[0054] When used for prokaryotic expression and purification of fusion proteins, this variant bifunctional protein tag also comprises a purification tag, a bifunctional tag, and a target protein. The bifunctional tag can be located at either the N-terminus or the C-terminus of the target protein. Prokaryotic expression vectors can include pET, pGEX, pMAL, and pCold vectors.

[0055] From a functional perspective, bifunctional protein tag variants can exhibit better performance when expressing certain specific types of proteins. For example, they can provide higher soluble expression levels when expressing membrane proteins containing a large number of hydrophobic regions; they can also provide higher thermal stability when expressing industrial enzymes that need to work under high temperature conditions.

[0056] Example 3 This example provides another variant of a bifunctional protein tag, whose amino acid sequence is 75% similar to that of SEQ ID NO: 1. This variant further expands the application range of bifunctional protein tags through a wider range of amino acid substitutions.

[0057] Specifically, based on Example 2, the variant further underwent amino acid substitutions at the following sites: position 76 was replaced by D to Q, position 77 was replaced by V to T, position 79 was replaced by A to E, position 80 was replaced by I to L, position 81 was replaced by I to F, position 82 was replaced by R to T, position 83 was replaced by I to L, position 84 was replaced by D to P, position 85 was replaced by V to I, position 86 was replaced by D to T, position 87 was replaced by D to N, position 88 was replaced by V to G, position 89 was replaced by N to L, position 94 was replaced by K to S, and position 97 was replaced by I to N.

[0058] These additional amino acid substitutions are primarily concentrated in the C-terminal region of the bifunctional protein tag. By optimizing the amino acid composition of this region, the compatibility of the tag with the target protein is further improved. In particular, the introduction of more polar amino acids enhances the interaction between the tag and the aqueous environment, improving the solubility of the fusion protein. By optimizing the distribution of charged amino acids, the isoelectric point of the tag is improved, making it more suitable for applications under different pH conditions.

[0059] When used for prokaryotic expression and purification of fusion proteins, this variant bifunctional protein tag also comprises a purification tag, a bifunctional tag, and a target protein. The bifunctional tag can be located at either the N-terminus or the C-terminus of the target protein. Prokaryotic expression vectors can include pET, pGEX, pMAL, and pCold vectors.

[0060] From the functional point of view, the dual-function protein tag variant has unique advantages in expressing some special types of proteins. For example, it can provide better pH stability when expressing proteins that need to work under extreme pH conditions; it can provide better surface compatibility when expressing proteins that need to interact with other proteins or small molecules.

[0061] Example 4 This example provides an application example of the dual-function protein tag in the fusion expression of poorly soluble proteins. The application selects a protein that is prone to form inclusion bodies in conventional expression systems, humanized single-chain antibody (scFv), as the target protein. Through fusion expression with the dual-function protein tag, the soluble expression of the protein is successfully realized.

[0062] First, the gene encoding humanized scFv is connected with the dual-function protein tag gene in Example 1 by PCR amplification and overlap extension PCR method to construct a fusion gene. The fusion gene is cloned into the pET-28a vector to form a fusion with the His tag sequence on the vector.

[0063] The recombinant plasmid is transformed into BL21 (DE3) E. coli expression strain and cultured in LB medium containing kanamycin overnight. The overnight culture is inoculated into fresh LB medium at a ratio of 1:100, and cultured at 37°C, 220 rpm until OD600 reaches 0.6. IPTG is added to a final concentration of 0.5 mM, and the culture is continued at 18°C for 16 hours to induce protein expression.

[0064] The bacterial cells are collected and resuspended in a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl and 20 mM imidazole. The bacterial cells are disrupted by ultrasonic wave, and the soluble proteins and insoluble components are separated by centrifugation at 12000 rpm for 30 minutes.

[0065] The supernatant is filtered through a 0.22 μm filter membrane and loaded onto a pre-equilibrated nickel ion affinity chromatography column. The target protein is eluted using an elution buffer containing 500 mM imidazole. The eluate containing the target protein is collected and analyzed for protein purity by SDS-PAGE.

[0066] The effectiveness of the dual-function protein tag in promoting the soluble expression of poorly soluble proteins can be judged by the amount of the target protein to be tested in soluble expression.

[0067] Example 5 This example provides an application example of the dual-function protein tag in improving the thermal stability of proteins. This example selects a heat-labile industrial enzyme, lipase, as the target protein, and successfully improves the thermal stability of the enzyme by fusion expression with the dual-function protein tag.

[0068] First, the gene encoding lipase was linked to the bifunctional protein tag variant gene in Example 2 by PCR amplification and overlap extension PCR to construct a fusion gene. The fusion gene was cloned into the pET-28a vector so that it formed a fusion with the His tag sequence on the vector.

[0069] Transform the recombinant plasmid into the BL21(DE3) E. coli expression strain and culture overnight in LB medium containing kanamycin. Inoculate the overnight culture into fresh LB medium at a ratio of 1:100 and incubate at 37°C, 220 rpm until the OD600 reaches 0.8. Add IPTG to a final concentration of 0.2 mM and continue incubation at 25°C for 12 hours to induce protein expression.

[0070] The cells were harvested and resuspended in a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 20 mM imidazole. The cells were disrupted by sonication and centrifuged at 12,000 rpm for 30 minutes to separate the soluble and insoluble components.

[0071] Filter the supernatant through a 0.22 μm filter and load onto a pre-equilibrated nickel affinity chromatography column. Elute the target protein using elution buffer containing 500 mM imidazole. Collect the eluate containing the target protein and analyze its purity by SDS-PAGE.

[0072] The thermal stability of the fusion protein and the original lipase can be measured by differential scanning calorimetry (DSC) to determine the effectiveness of the bifunctional protein tag in improving the thermal stability of the protein.

[0073] Example 6 This example provides an application example of a bifunctional protein tag for increasing protein expression. This example selected a humanized growth factor, a protein with low expression levels in E. coli, as the target protein. By fusion expression with the bifunctional protein tag, the protein's expression was successfully increased.

[0074] First, the gene encoding the humanized growth factor was linked to the bifunctional protein tag variant gene described in Example 3 by PCR amplification and overlap extension PCR to construct a fusion gene. The fusion gene was cloned into the pET-28a vector so that it fused to the His tag sequence on the vector.

[0075] The recombinant plasmid was transformed into BL21(DE3) E. coli expression strain, and cultured in LB medium containing kanamycin overnight. The overnight culture was inoculated into fresh LB medium at a ratio of 1:100, and cultured at 37°C, 220 rpm until the OD600 reached 0.7. IPTG was added to a final concentration of 0.1 mM, and the culture was continued at 20°C for 14 hours to induce protein expression.

[0076] The bacterial cells were collected and resuspended in a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 20 mM imidazole. The bacterial cells were disrupted by ultrasonic waves, and the soluble proteins and insoluble components were separated by centrifugation at 12,000 rpm for 30 minutes.

[0077] After filtering the supernatant through a 0.22 μm filter, it was loaded onto a pre-equilibrated nickel ion affinity chromatography column. The target protein was eluted using an elution buffer containing 500 mM imidazole. The eluate containing the target protein was collected and analyzed for protein purity and expression level by SDS-PAGE.

[0078] The biological activity of the purified protein can be determined by comparing the expression level with that of a humanized growth factor without a bifunctional protein tag, which can be used to demonstrate the effectiveness of the bifunctional protein tag in improving protein expression.

[0079] The advantages of the present application are illustrated below through specific experiments and corresponding effects: 1. Design and structure verification of nCutA tag The sequences with high correlation to CUTA protein were screened from the UniPort database and aligned, and the secondary structure was labeled, as shown in Figure 1 The basic structure of CUTA consists of multiple alpha helices and beta parallel structures, including beta1-alpha1-beta2-beta3-eta1-alpha2-beta4-alpha3, wherein the conserved region is located inside the secondary structure, and the variable region is basically located at the position of secondary structure transformation, i.e., at the alpha / beta junction region, which can be used for protein modification based on this structure.

[0080] The CutA protein derived from Pyrococcus horikoshii is named PHCUTA, and the nCUTA monomer three-dimensional structure is predicted using AlphaFold3, as shown in Figure 2 Figure 2 The left side of the A in the middle is the monomer structure of CUTA, Figure 2 The left side of the B in the middle is the monomer structure of PHCUTA, and the middle column is the predicted structure of nCUTA. Through structure fitting, the results show that the monomer structure is basically consistent with CUTA, and nCUTA has similar structure.

[0081] ​The protein hydrophobicity of CUTA and nCUTA was analyzed using an online tool. The horizontal axis represents the amino acid site and the vertical axis represents the amino acid hydrophobicity score. The more hydrophilic the amino acid, the higher the value. Conversely, the more hydrophilic the amino acid, the lower the value. The results are as follows: Figure 3 As shown in the figure, compared with CUTA, nCUTA has more amino acids below the horizontal axis, which means it is more hydrophilic.

[0082] Using the protein solubility prediction tool SoluProt, we input the protein sequences of these three vectors in FASTA format into a specified region and returned prediction results. The results are shown in Table 1. pCold II-FT scored 0.454. A score below 0.5 indicates insoluble expression. In practice, the purified FT protein yield is approximately 5 mg / 4 L, resulting in an extremely low purification yield. pCold II-nCUTA-FT scored 0.701. A solubility score above 0.5 indicates soluble expression in E. coli. pCold II-NusA-FT achieved the highest score, 0.920, indicating extreme solubility.

[0083] Table 1 Prediction of FT protein solubility after expression

[0084] 2. Expression and purification of nCutA-T4 DNA PolA fusion protein Using primer design, the T4 DNA PolA and CutA-T4 DNA PolA DNA fragments were double-digested with EcoRI and BamHI, respectively, and pCold II was double-digested. The double-digested T4 DNA PolA and CutA-T4 DNA PolA DNA fragments were then ligated with the double-digested pCold II fragment using T4 DNA ligase overnight in an ice-water mixture. The next day, the ligation products were transformed into DH5α competent cells and incubated overnight at 37°C. The following day, colony PCR was performed to screen for viable clones. Positive clones were selected and incubated overnight at 37°C, 220 rpm, for plasmid DNA extraction.

[0085] The extracted plasmid DNA pCold-T4 DNA PolA and pCold-CutA T4 DNA PolA were transformed into Rosetta competent cells. After overnight culture at 37°C and 220 rpm, the cells were transferred to 600 ml of LB medium and cultured at 37°C until OD600 was ≈ 0.6. IPTG was then added to a final concentration of 0.5 mM and cultured at 16°C overnight.

[0086] The overnight culture was centrifuged at 8000 rpm for 5 minutes to collect the cells. The resulting cells were resuspended in Buffer A at 30 ml / g wet weight and disrupted for 560 minutes at 200W, with pulses of 5 seconds and rests of 10 seconds. The supernatant was then centrifuged at 12000 rpm for 30 minutes. The supernatant was filtered through a 0.22 μm filter and purified using a nickel affinity chromatography column.

[0087] The buffer used was as follows: Buffer A: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 20 mM imidazole. Buffer B: 20 ​​mM Tris-HCl (pH 8.0), 300 mM NaCl, 500 mM imidazole.

[0088] The specific steps for affinity chromatography are as follows: Purification instrument inspection and cleaning: Turn on the AKTA PURE protein purifier and inspect the waste container, pump head, water bottle, and autosampler. Set the system and line pressure to 0.5 MPa. Flush the system with ultrapure water by clicking A Wash, B Wash, and System Wash to flush the pump and lines. Set the column direction to downward, close the restrictor, and set the flow rate to 1 ml / min at 50% Buffer B. Observe the changes in conductivity and UV absorbance until the conductivity reaches zero and the UV absorbance stabilizes before loading the sample. When the UV value rises rapidly, collect the flow-through sample (after column loading). After the UV value decreases and stabilizes again, run a gradient (slowly increasing the Buffer B content to 100% over 10 minutes). After the UV value rises rapidly again, begin collecting the purified protein sample and stop collecting the sample until the UV value decreases and stabilizes again. Then, perform SDS-PAGE electrophoresis on the pre-induction, post-induction, pre-column, post-column, and purified protein samples. Finally, Coomassie brilliant blue staining and Image J analysis were performed.

[0089] The results are as follows Figure 4 As shown, in Figure 4 In Figure A, the protein expression level at the target band in lane 2 (after induction) is significantly higher than that in lane 1 (before induction), indicating that T4 DNA polymerase is successfully induced. The target protein band in lane 4 (flow-through) is significantly reduced compared to lane 3 (before loading), indicating that T4 protein is successfully bound to the chromatography column. Lanes 5-12 indicate that the target protein is successfully eluted. Figure 4 In Figure B, the protein expression level at the target band in lane 2 (after induction) was significantly increased compared with lane 1 (before induction), indicating that CUTA-T4 DNA polymerase was successfully induced. The target protein band in lane 4 (flowthrough) was significantly reduced compared with lane 3 (before loading), indicating that CUTA-T4 protein was successfully bound to the chromatography column. Lanes 5-14 showed that the target protein was successfully eluted.

[0090] 3. T4 DNA polymerase thermal stability test T4 DNA polymerase is a monomeric enzyme composed of a single polypeptide chain. It has multiple structural domains, including those responsible for DNA binding and the catalytic active center. These domains work synergistically to enable T4 DNA polymerase to specifically recognize and bind to DNA templates and perform efficient DNA synthesis. It is a template-dependent DNA polymerase that catalyzes DNA synthesis in the 5'→3' direction in the presence of a single-stranded DNA template and its paired primer.

[0091] Use complementary primers and template: Template (SEQ ID NO: 2): 5'-ATACATAGATACATAGACTGGCCGTCGTTTTAC-3'; Primer (SEQ ID NO: 3): 5′-GTAAAACGACGGCCAGT-3′; Prepare the annealing system (100 μl): 40 μl of sterile water, 20 μl of Template (50 μM), 20 μl of Primer (50 μM), and 20 μl of Annealing Buffer for DNA Oligos (5×). Add all reagents in the order listed above and mix thoroughly. Perform the annealing reaction using a PCR instrument: heat at 95°C for 2 minutes to fully denature the oligos, then decrease the temperature by 0.1°C every 8 seconds until the temperature reaches 25°C. Finally, store at 4°C and store at -20°C.

[0092] Activity assay reaction system (20 μl): 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, 125 μM dNTP Mix, 0.5 μM Template / Primer.

[0093] The reaction was performed using a PCR instrument, initially incubating at 12°C for 15 minutes, followed by incubation at 4°C for 5 minutes, and terminated by incubation at 75°C for 20 minutes. A 10 μl aliquot of the reaction product was added to 1.1 μl of 10× DNA loading buffer, and then subjected to electrophoresis on a 15% native polyacrylamide gel. Electrophoresis was performed at 180 V for 60 minutes. The gel was stained with Gel-Red (10,000×) for 15 minutes at room temperature, and then photographed using a gel imaging system.

[0094] After heat treatment of T4 and CUTA-T4, the reaction was carried out and the results were analyzed by agarose gel electrophoresis and Image J. Figure 5 In Figure A, lanes 3 and 4 are the 53.5℃ treatment group, where more CUTA-T4 products were observed on the right side, with a significant difference (p<0.0001); lanes 5 and 6 are the 53.9℃ treatment group, where more CUTA-T4 products were observed on the right side, with a significant difference (p<0.01); lanes 7 and 8 are the 55.0℃ treatment group, where more CUTA-T4 products were observed on the right side, with a significant difference (p<0.0001); lanes 9 and 10 are the 56.0℃ treatment group, where more CUTA-T4 products were observed on the right side, with a significant difference (p<0.0001); lanes 11 and 12 are the 56.0℃ treatment group, where more Lanes 11 and 12 represent the 57.3°C treatment group, where there was no significant difference between the two groups. Lanes 13 and 14 represent the 58.5°C treatment group, where more CUTA-T4 products were observed on the right side, a significant difference (p<0.001). Lanes 15 and 16 represent the 57.3°C treatment group, where there was a significant difference (p<0.0001). Lanes 17 and 18 represent the 60.0°C treatment group, where there was no significant difference. The results show that CUTA-T4 DNA polymerase has higher activity than T4 DNA polymerase after treatment at the same temperature, indicating that the CUTA tag significantly improves the thermal stability of T4 DNA polymerase.

[0095] 4. nCUTA improves the solubility of inclusion body protein FT Three plasmid DNAs, pCold II-FT, pCold II-nCUTA-FT, and pCold II-NusA-FT, were constructed by double enzyme digestion and transformed. Competent cells were expressed using Rosetta 2 (DE3). After small shaking, large shaking, and induction, the bacteria were collected. The collected bacteria were resuspended in Buffer A at 30 ml / g according to their mass, and 1 ml was taken for packaging. One tube was taken for ultrasonic disruption of bacteria, 200 w, pulse 5 sec, interval 10 sec, 5 min. Then centrifuged at 12000 rpm for 10 min, part of the supernatant was taken for sample preparation, and the remaining supernatant was added with PMSF, DTT, etc. and placed at 4°C for use. After all the supernatant was taken out, the precipitate was resuspended in 1 ml Buffer A, and then the samples before and after induction, the supernatant after disruption of bacteria, and the precipitate after disruption of bacteria were subjected to SDS-PAGE electrophoresis, and WB analysis was performed by Coomassie Brilliant Blue staining. Then, the changes in FT protein solubility were analyzed by Image J. The results are shown in the figure. Figure 6As shown in the results, compared with the control FT group, the nCUTA tag significantly increased the relative expression of the target protein FT in the supernatant (p<0.0001), which was 3.6 times the relative expression of FT; the NusA tag significantly increased the relative expression of the target protein FT in the supernatant (p<0.0001), which was 9.6 times the relative expression of FT. In terms of absolute expression, the nCUTA tag significantly increased the absolute expression of the target protein FT in the supernatant, which was 3.6 times the absolute expression of FT; the NusA tag significantly increased the absolute expression of the target protein FT in the supernatant, which was 4.3 times the absolute expression of FT.

[0096] 5. Effect of nCUTA tag on FTase activity High-performance liquid chromatography (HPLC) was used to characterize the FTase-catalyzed transfer of guanosine diphosphate-L-fucose (GDP-L-Fucose) to N-acetyllactosamine (LacNAc), yielding the product α-L-fucosyl-(1→2)-β-D-galactosyl-(1→4)-N-acetylglucosamine (Fucα1-2Galβ1-4GlcNAc) and the byproduct guanosine diphosphate (GDP). The products of the FTase-catalyzed reaction were separated using the separation capabilities of HPLC. The products were qualitatively and quantitatively analyzed based on their retention time and peak area on the chromatographic column, thereby determining the amount of product generated from the substrate catalyzed by the FTase and measuring the relative activity of the FTase.

[0097] Chromatographic conditions: Aglient C18 4.6 × 250 mm, 5 μm column, detection wavelength 254 nm, column temperature 25.0°C, flow rate 1.1 ml / min, ratio: 90% A + 10% B. Injection volume 100 μl.

[0098] Mobile phase: Phase A: TBAB, Phase B: acetonitrile.

[0099] The activity detection reaction system (100 μl) is shown in Table 2.

[0100] Table 2 FT activity detection system

[0101] Sample pretreatment and enzyme activity determination: Add the reactants in sequence according to the following system, adding the enzyme to be tested last. For the control, no enzyme was added. Pretreat the three proteins at 36°C, 40°C, 45°C, and 50°C for 10 minutes, respectively, before adding the prepared system. Mix the samples thoroughly and incubate them on a shaker at 37°C and 200 rpm for 30 minutes. Centrifuge them at 4°C and 12,000 rpm for 10 minutes. Pipette 50 μl of the supernatant into a liquid phase vial containing 950 μl of ultrapure water and mix thoroughly. Place the sample in the loading rack, set up the batch file, and click Start. Determine the substrate in the sample reaction based on the substrate retention time of the control. Calculate the product content based on the substrate peak area, and then calculate the relative FTase activity.

[0102] The results are as follows Figure 7 and Figure 8 As shown, after pretreatment at 36°C, all substrates in the FT, nCUTA-FT, and NusA-FT groups reacted completely (peak elution time: 20 min), generating the target product (peak elution time: 22 min), indicating an activity of 100%. After treatment at other temperatures, differences were observed among the FT, nCUTA-FT, and NusA-FT groups. After pretreatment at 50°C, substrates in the FT and NusA-FT groups remained largely unreacted, with the exception of a slight decrease in substrate in the nCUTA-FT group.

[0103] Figure 7 This is a composite diagram of the HPLC results of FT-catalyzed guanosine diphosphate-L-fucose (GDP-L-Fucose) and N-acetyllactosamine (LacNAc). The substrate peak (about 20 min) and product peak (about 22 min) can be seen in the figure. The first column from the left is the control group FT, the middle is nCUTA-FT, and the rightmost is NusA-FT; from top to bottom, they are 36°C, 40°C, 45°C, and 50°C, respectively.

[0104] like Figure 8 As shown, after pretreatment at 40℃, the relative activity of the nCUTA-FT group was significantly different compared with the FT group ( p <0.01), NusA-FT showed significant differences in activity ( p <0.0001); after pretreatment at 45℃, the relative activity of nCUTA-FT group had no significant difference compared with FT group, while NusA-FT had significant difference in activity ( p <0.0001); after pretreatment at 50℃, the relative activity of the nCUTA-FT group was significantly different from that of the FT group ( p<0.0001), and NusA-FT showed no significant difference in activity. These results suggest that the nCUTA tag enhances the thermal stability of the FT protein, resulting in differences in activity under different temperature treatments. The relatively low activity of the NusA group may be due to the large molecular weight of the tag, which affects activity.

[0105] The above results demonstrate that the nCUTA tag can improve the solubility of the target protein FT. Furthermore, FT activity tests show that, compared with the control group, nCUTA-FT exhibits significantly higher activity at different temperatures, indicating that nCUTA enhances the thermal stability of the FT protein. The nCUTA tag has both solubility-enhancing and thermal stability-enhancing properties.

[0106] In summary, while novel tags derived through rational design and modification of natural proteins (such as hydrophilic engineered peptides) show promise, bifunctional tags combining high solubility, high thermal stability, and a low molecular weight (<15 kDa) remain unavailable. In particular, for the expression of industrial enzymes prone to inclusion body formation, such as fucosyltransferase (FT), traditional tags cannot simultaneously address the dual bottlenecks of low solubility and high-temperature inactivation. Based on these considerations, the present invention utilizes multiple sequence alignment to modify the thermostable CutA protein, further enhancing its hydrophilicity without altering its protein structure. This protein tag, designed by rationally modifying the hydrophobic regions of the native CutA protein, significantly enhances the solubility and thermal stability of the fusion protein in Escherichia coli expression systems, making it suitable for the functional expression and industrial application of inclusion body proteins. By placing the CutA bifunctional protein tag at either the N-terminus or the C-terminus of the fusion protein, the present invention significantly improves its thermal stability and solubility. 3. Compared with traditional protein tags that can only improve the thermal stability of proteins within 15Kd, the bifunctional protein tag provided by the present invention can improve the thermal stability of proteins above 100Kd and has a wider range of applications.

[0107] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A bifunctional protein tag for improving the thermal stability and solubility of a recombinant protein, characterized in that: These include proteins such as: a) a protein with the amino acid sequence shown in SEQ ID NO: 1; b) a protein having an amino acid sequence that is greater than 70% to 90% identical to SEQ ID NO: 1 after substitution and / or deletion and / or addition of amino acid residues to the sequence shown in SEQ ID NO: 1 and having the same function as a); c) A protein formed by splicing of a) and / or b) and having the same function as a).

2. The bifunctional protein tag for improving the thermal stability and solubility of a recombinant protein according to claim 1, characterized in that: This bifunctional protein tag is composed of multiple α-helices and β-pleated sheets assembled into a stable trimer structure according to the topological arrangement of β1-α1-β2-β3-η1-α2-β4-α3; among them, the β-pleated sheets are arranged in a parallel manner and stabilized by a hydrogen bond network, and the α-helices form stable helical bundles through side chain hydrophobic interactions and interchain hydrogen bonds.

3. The bifunctional protein tag for improving the thermal stability and solubility of a recombinant protein according to claim 1, wherein: Sequence mutations with 70% to 90% identity to SEQ ID NO: 1 include: The amino acid sequence at position 27 is I / A, the amino acid sequence at position 30 is A / F / V, the amino acid sequence at position 33 is R / F / V / I, the amino acid sequence at position 38 is G / F / I / L, the amino acid sequence at position 40 is W / R / Y, the amino acid sequence at position 44 is E / K, the amino acid sequence at position 45 is I / V / L, the amino acid sequence at position 46 is E / V, the amino acid sequence at position 52 is G / A / Q, the amino acid sequence at position 60 is D / E / S, the amino acid sequence at position 61 is L / K / A / H, the amino acid sequence at position 62 is W / E / Q, the amino acid sequence at position 64 is E / A, the amino acid sequence at position 66 is K / L, the amino acid sequence at position 68 is R / E / C, the amino acid sequence at position 69 is I / L / V, the amino acid sequence at position 70 is K / R, and the amino acid sequence at position 71 is The amino acid sequence at position 1 is E / K / S, the amino acid sequence at position 72 is L / H, the amino acid sequence at position 76 is D / E / Q, the amino acid sequence at position 77 is V / T, the amino acid sequence at position 79 is A / E / C, the amino acid sequence at position 80 is I / L, the amino acid sequence at position 81 is I / F / V / L, the amino acid sequence at position 82 is R / T / A / V, the amino acid sequence at position 83 is I / L, the amino acid sequence at position 84 is D / P, the amino acid sequence at position 85 is V / I, the amino acid sequence at position 86 is D / E / T / A, the amino acid sequence at position 87 is D / N / E / H / Q, the amino acid sequence at position 88 is V / G, the amino acid sequence at position 89 is N / L / D, the amino acid sequence at position 94 is K / N / D / S, and the amino acid sequence at position 97 is I / N / R.

4. Use of the bifunctional protein tag for improving the thermal stability and solubility of a recombinant protein according to any one of claims 1 to 3 in soluble protein expression.

5. A fusion protein, characterized in that The invention comprises at least a target protein and a bifunctional protein tag for improving the thermal stability and solubility of a recombinant protein according to any one of claims 1 to 3, wherein the bifunctional protein tag for improving the thermal stability and solubility of a recombinant protein is at the N-terminus or C-terminus of the target protein.

6. The fusion protein according to claim 5, characterized in that A protease recognition site and / or a purification tag is also included between the target protein and the bifunctional protein tag for improving the thermal stability and solubility of the recombinant protein.

7. An expression vector, characterized in that It is a prokaryotic expression vector containing the fusion protein or its encoding gene according to claim 5 or 6.

8. The expression vector according to claim 7, characterized in that The prokaryotic expression vector can be selected from the pET series vectors, pGEX series vectors, pMAL series vectors or pCold series vectors.

9. A host cell, characterized in that The host cell comprises the expression vector according to claim 7 or 8, and is capable of expressing the fusion protein.

10. Use of the fusion protein according to claim 5 or 6, the expression vector according to claim 7 or 8, or the host cell according to claim 9 in protein expression and purification.

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