A temperature-sensitive and mmp-9 enzyme-responsive fusion protein, and a preparation method and application thereof

Abstract

The application discloses a temperature-sensitive and MMP-9 enzyme-responsive fusion protein and a preparation method and application thereof, and belongs to the fields of biological medicine and protein engineering. The fusion protein comprises, from an N terminus to a C terminus, an elastin-like polypeptide, a matrix metalloproteinase-9 and a targeted binding domain in sequence. The amino acid sequence of the elastin-like polypeptide is shown as SEQ ID NO. 3. The amino acid sequence of the matrix metalloproteinase-9 is shown as SEQ ID NO. 2. The amino acid sequence of the targeted binding domain is shown as SEQ ID NO. 1. The fusion protein has the characteristics of temperature-sensitive phase transition and MMP-9 responsiveness, has good binding activity with PTPsigma, and has a good application prospect in the field of drug delivery.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and protein engineering, and in particular to a temperature-sensitive and MMP-9 enzyme-responsive fusion protein, its preparation method, and its applications. Background Technology

[0002] Elastin-like peptides (ELPs) are a class of synthetically produced thermosensitive peptides composed of (VPGXG). n The pentapeptide repeat sequence is composed of a phase transition temperature (T0). t It is soluble at T. t Reversible liquid-liquid phase separation (LLPS) occurs, forming protein-rich aggregates. This property makes it a potential candidate for applications in drug delivery, tissue engineering, and other fields. However, natural ELPs lack active targeting capabilities, have limited functionality, and exhibit poor stability and controllability.

[0003] In recent years, deep learning-assisted protein design technologies (such as RFDiffusion and ProteinMPNN) have made it possible to design novel protein domains with specific binding functions from scratch. However, there are currently no reports of fusing such designed domains with ELP to construct proteins with triple functions of target recognition, temperature phase transition and enzyme response. Summary of the Invention

[0004] The purpose of this invention is to provide a temperature-sensitive and MMP-9 enzyme-responsive fusion protein SEP, its preparation method, and its applications, in order to solve the problems existing in the prior art. This fusion protein undergoes LLPS at specific concentrations and temperatures, and its phase transition temperature can be adjusted by protein concentration, ionic strength, and pH. It has the characteristics of temperature-sensitive phase transition and MMP-9 responsiveness, and has good binding activity with PTPσ, showing good application prospects in the field of drug delivery.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a fusion protein, which comprises, from the N-terminus to the C-terminus, an elastin-like polypeptide, a matrix metalloproteinase-9 cleavage site, and a target-binding domain. The amino acid sequence of the elastin-like polypeptide is shown in SEQ ID NO.3; the amino acid sequence of the matrix metalloproteinase-9 cleavage site is shown in SEQ ID NO.2; and the amino acid sequence of the target-binding domain is shown in SEQ ID NO.1.

[0006] Preferably, the matrix metalloproteinase-9 cleavage site includes a matrix metalloproteinase recognition and cleavage site, and the targeting binding domain is a binding protein capable of specifically binding to the Ig1-2 domain of protein tyrosine phosphatase σ.

[0007] The present invention also provides a nucleic acid molecule encoding the fusion protein described herein.

[0008] Preferably, the sequence of the nucleic acid molecule is as shown in SEQ ID NO.4.

[0009] The present invention also provides a recombinant vector comprising the aforementioned nucleic acid molecule.

[0010] The present invention also provides a recombinant bacterium comprising the aforementioned recombinant vector.

[0011] The present invention also provides a method for constructing the fusion protein, comprising the following steps: S1. Construct a prokaryotic expression vector containing the gene encoding the fusion protein, and construct a recombinant vector; S2. Transform the recombinant vector into Escherichia coli host cells to construct recombinant bacteria; S3. The recombinant bacteria are induced to express at 16-20℃ and 0.1-0.3 mM IPTG for 12-48 h; most preferably, the IPTG induction conditions are: 16 ℃ and 0.2 mM for 36 h. S4: Centrifuge to collect bacterial cells, treat with hypertonic buffer on ice, then lyse with hypotonic buffer, centrifuge to collect supernatant to obtain crude protein solution; S5. The crude protein solution is purified by thermal cycling phase change method. The target protein is precipitated under high temperature and high salt conditions, the precipitate is collected by centrifugation, and the target protein is reconstituted under low temperature and low salt conditions. The above steps are repeated 2-4 times to obtain the purified fusion protein.

[0012] Preferably, the thermal cycling phase transition method includes the following steps: adding NaCl to the crude protein solution to a final concentration of 2M, incubating at 37°C, centrifuging to precipitate the target protein, resuspending the precipitate in pre-cooled PBS, collecting the supernatant by low-temperature centrifugation at 4°C, repeating the thermal cycling purification three times, and concentrating the purified fusion protein using a 5 kDa ultrafiltration tube.

[0013] The present invention also provides a method for detecting the matrix metalloproteinase-9 responsiveness of the fusion protein in vitro, comprising the following steps: The fusion protein was dissolved in an enzyme digestion reaction buffer, and then incubated with matrix metalloproteinase-9. The protein changes at different incubation time points from 0 to 24 h were detected by SDS-PAGE electrophoresis. The changes in protein bands were used to determine whether the fusion protein had matrix metalloproteinase-9 responsiveness.

[0014] The present invention also provides the use of the fusion protein in any of the following: (1) Application in the preparation of temperature-sensitive protein assemblies; (2) Application in the preparation of matrix metalloproteinase-9 responsive protein deassembly and assembly system; (3) Application in the preparation of targeted drug delivery vectors.

[0015] The present invention discloses the following technical effects: (1) This invention integrates the target domain, MMP-9 restriction site and ELP temperature-sensitive core designed by deep learning into a single protein through genetic engineering, and for the first time realizes the synergistic effect of the triple functions of target recognition, enzyme response and temperature-induced phase transition in one molecule.

[0016] (2) The fusion protein of the present invention retains the thermosensitive properties of ELP. Its phase transition temperature can be flexibly controlled by protein concentration, salt concentration and pH. Variable temperature circular dichroism spectroscopy confirms that its phase transition is accompanied by orderly changes in secondary structure, and it can be assembled in multiple stages.

[0017] (3) The fusion protein of the present invention exhibits unique multi-level assembly behavior, from polydisperse aggregates at low temperatures to near-T0... m From uniform nanoparticles to micron-sized droplets at high temperatures, this provides a new strategy for constructing protein structures of different scales.

[0018] (4) By introducing the MMP-9 restriction site, the present invention enables the fusion protein to be cleaved and disassembled in a specific pathological microenvironment. After restriction, the Binder domain separates from the ELP core, which can realize the release of the targeted function.

[0019] (5) Simple preparation process: The “thermal cycling” purification method established by utilizing the phase change characteristics of ELP does not require expensive affinity chromatography. The steps are simple, the cost is low, and it is easy to scale up. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The results show the molecular docking between PTPσ and Binder; Figure 2 The pET-22b(+)-SEP plasmid map; Figure 3 SDS-PAGE image of purified SEP fusion protein; Figure 4 The results of optimizing the SEP fusion protein expression inducer concentration (a), induction time (b), and induction temperature (c) are shown. Figure 5 The diagram shows the phase transition of the temperature-induced SEP fusion protein; (a) liquid-liquid phase separation of SEP, (b) optical microscope image of SEP phase separation; Figure 6 The circular dichroism chromatogram of SEP at 221 nm at varying temperatures; Figure 7 SDS-PAGE images of SEP fusion protein digested at different times (0, 0.5, 1, 2, 4, 8, 16, 24 h); M represents the standard protein molecule; Figure 8 The turbidity-temperature curve of the SEP fusion protein. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Example 1: Sequence design and gene synthesis of SEP (Signaling Elastin-like Protein) fusion protein S1. The Ig1-2 domain (PDB ID: 2YD3) of protein tyrosine phosphatase σ (PTPσ) was used as the target site, and K42, K43, K45, K46, R71 and R74 on its surface were defined as key binding hot spots.

[0028] S2. Using the deep learning protein design tool RFDiffusion, K42, K43, K45, K46, R71, and R74 were set as binding hotspot residues, the number of amino acids was limited to ≤50, the number of outputs was 100, and finally 100 candidate backbones were generated, which were then optimized by ProteinMPNN.

[0029] S3. Molecular docking screening was performed using HADDOCK, combined with IUPred2A prediction of hydrophilicity and hydrophobicity, ultimately identifying hydrophilic targeting binding domain (Binder) sequences (see...). Figure 1 ).

[0030] S4. The ELP core sequence (amino acid sequence as shown in SEQ ID NO.3), the matrix metalloproteinase-9 (MMP-9) restriction site (amino acid sequence as shown in SEQ ID NO.2), and the Binder sequence (amino acid sequence as shown in SEQ ID NO.1) are fused in sequence from N-terminus to C-terminus to obtain the SEP protein-coding gene (nucleotide sequence as shown in SEQ ID NO.4). This gene is synthesized and cloned into the BamHI and XhoI sites of the pET-22b(+) vector to obtain the recombinant plasmid pET-22b(+)-SEP (see diagram). Figure 2 ).

[0031] Example 2: Expression and purification of SEP fusion protein S1. The recombinant plasmid pET-22b(+)-SEP was transformed into E. coli BL21(DE3) competent cells and plated on LB agar plates containing ampicillin. Single colonies were picked and inoculated into LB medium and activated overnight at 37°C and 220 rpm. The cells were then transferred to TB medium at a volume ratio of 1:100 and cultured at 37°C with shaking until OD (out of control) was reached. 600 =0.6-0.8, add IPTG to a final concentration of 0.2 mM, and induce expression at 16℃ and 220 rpm for 36 h.

[0032] S2. Centrifuge to collect bacterial cells. Add 100 mL of pre-cooled hypertonic buffer (20% sucrose, 30 mM Tris-HCl, 1 mM EDTA) per gram of bacterial cells, incubate on ice for 20 min, centrifuge and discard the supernatant. Quickly add an equal volume of pre-cooled hypotonic buffer (5 mM MgCl2), vortex to resuspend, incubate on ice for 30 min, centrifuge and collect the supernatant to obtain the crude protein solution.

[0033] S3. Add NaCl to the crude protein solution to a final concentration of 2 M, incubate in a 37°C water bath for 10 min, centrifuge at 13000×g at 37°C for 10 min, and discard the supernatant. Resuspend the precipitate in pre-cooled PBS, centrifuge at 10000 rpm at 4°C for 10 min, and collect the supernatant. Repeat this "thermal cycling" operation 3 times, and finally concentrate using a 5 kDa ultrafiltration tube to obtain the purified SEP fusion protein (see...). Figure 3 ).

[0034] Example 3: Optimization of SEP fusion protein expression conditions (1) Optimization of inducer concentration The difference from step S1 in Example 2 is that the induction temperature was fixed at 20 °C, the induction time was 24 h, and the final concentration of the inducer was changed to 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mM respectively; all other conditions were the same as in Example 2. After expression and purification, the yield of the target protein was measured and calculated.

[0035] (2) Optimization of induction temperature The difference from step S1 in Example 2 is that the final concentration of the inducer was fixed at 0.2 mM, the induction time was 24 h, and the induction temperature was changed to 16, 20, 24, 30, and 37 °C. All other conditions were the same as in Example 2. After expression and purification, the yield of the target protein was measured and calculated.

[0036] (3) Optimization of induction time The difference from step S1 in Example 2 is that the final concentration of the inducer was fixed at 0.2 mM, the induction temperature was 20 °C, and the induction time was changed to 12, 18, 24, 36, and 48 h; all other conditions were the same as in Example 2. After expression and purification, the yield of the target protein was measured and calculated.

[0037] like Figure 4 As shown, the optimal induction conditions are: IPTG concentration of 0.2 mM, induction temperature of 16 ℃, and induction time of 36 h.

[0038] Example 4: Characterization of the thermosensitive phase transition behavior of the SEP fusion protein Prepare a 0.2 mg / mL SEP fusion protein solution and measure its turbidity (OD) at different temperatures. 600) and particle size distribution. The results show that the phase transition temperature (T) t ) is approximately 34℃ (see Figure 8 Dynamic light scattering (DLS) measurements showed that the particle size distribution was uneven at 4°C; uniform nanoparticles formed at 20°C; and uniform micron-sized droplets formed at 37°C (see...). Figure 5 ).

[0039] The temperature-dependent secondary structure of SEP was investigated using circular dichroism spectroscopy (CD). The detection wavelength was set to 221 nm, the temperature scan range was 10–60 °C, and the temperature was continuously increased at a linear rate of 1 °C / min. CD signal values ​​were acquired every 2 °C increase, and the equilibration time at each temperature point was set to 2 min. The change in molar ellipticity at 221 nm was monitored using variable-temperature CD, and the thermal transition temperature T was calculated through fitting. m It is 20.8℃ (see) Figure 6 ).

[0040] Example 5: Validation of MMP-9 responsiveness of SEP fusion protein The SEP fusion protein was dissolved in enzyme digestion buffer to prepare a 0.6 mg / mL solution. Human recombinant MMP-9 was added to a final concentration of 100 nM, and the mixture was incubated at 37 °C. At time points of 0, 0.5, 1, 2, 4, 8, 16, and 24 h, 40 μL of the reaction solution was collected, and immediately 10 μL of 5× protein loading buffer was added. The mixture was then inactivated by denaturation at 80 °C, and SDS-PAGE electrophoresis was performed to detect changes in protein bands.

[0041] The results are as follows Figure 7 As shown, the SEP fusion protein was gradually and effectively cleaved over time after protease treatment, releasing the target protein band of the expected size, and no significant degradation was observed within 24 hours. This demonstrates that the present invention, by modifying ELP, endows it with the ability to specifically recognize and respond to enzymes characteristic of the disease microenvironment (such as MMP-9).

[0042] The sequence involved in this invention: Binder sequence (SEQ ID NO:1): EEERRREEEEKERREREREEEEERRRKA.

[0043] Amino acid sequence of the MMP-9 restriction site (SEQ ID NO:2): PVGLIGGGSG.

[0044] ELP core sequence (SEQ ID NO:3): MSKGPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGHGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGWP。

[0045] Coding gene sequence of SEP protein (SEQ ID NO:4):

[0046] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises, from N-terminus to C-terminus, an elastin-like polypeptide, a matrix metalloproteinase-9 cleavage site, and a target-binding domain. The amino acid sequence of the elastin-like polypeptide is shown in SEQ ID NO.3; the amino acid sequence of the matrix metalloproteinase-9 cleavage site is shown in SEQ ID NO.2; and the amino acid sequence of the target-binding domain is shown in SEQ ID NO.

1.

2. The fusion protein as described in claim 1, characterized in that, The matrix metalloproteinase-9 cleavage site includes matrix metalloproteinase recognition and cleavage sites, and the target binding domain is a binding protein capable of specifically binding to the Ig1-2 domain of protein tyrosine phosphatase σ.

3. A nucleic acid molecule encoding the fusion protein of claim 1 or 2.

4. The nucleic acid molecule as described in claim 3, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID NO.

4.

5. A recombinant vector, characterized in that, It includes the nucleic acid molecules described in claim 3 or 4.

6. A recombinant bacterium, characterized in that, It includes the recombinant vector as described in claim 5.

7. A method for constructing the fusion protein according to claim 1 or 2, characterized in that, Includes the following steps: S1. Construct a prokaryotic expression vector containing the gene encoding the fusion protein, and construct a recombinant vector; S2. Transform the recombinant vector into Escherichia coli host cells to construct recombinant bacteria; S3. The recombinant bacteria were induced to express at 16-20℃ and 0.1-0.3 mM IPTG for 12-48 h. S4: Centrifuge to collect bacterial cells, treat with hypertonic buffer on ice, then lyse with hypotonic buffer, centrifuge to collect supernatant to obtain crude protein solution; S5. The crude protein solution is purified by thermal cycling phase change method. The target protein is precipitated under high temperature and high salt conditions, the precipitate is collected by centrifugation, and the target protein is reconstituted under low temperature and low salt conditions. The above steps are repeated 2-4 times to obtain the purified fusion protein.

8. The construction method as described in claim 7, characterized in that, The thermal cycling phase transition method includes the following steps: adding NaCl to the crude protein solution to a final concentration of 2 M, incubating at 37°C, centrifuging to precipitate the target protein, resuspending the precipitate in pre-cooled PBS, collecting the supernatant by low-temperature centrifugation at 4°C, repeating the thermal cycling purification three times, and concentrating the purified fusion protein using a 5 kDa ultrafiltration tube.

9. A method for in vitro detection of the matrix metalloproteinase-9 responsiveness of the fusion protein of claim 1, characterized in that, Includes the following steps: The fusion protein was dissolved in an enzyme digestion reaction buffer, and then incubated with matrix metalloproteinase-9. The protein changes at different incubation time points from 0 to 24 h were detected by SDS-PAGE electrophoresis. The changes in protein bands were used to determine whether the fusion protein had matrix metalloproteinase-9 responsiveness.

10. The use of the fusion protein as described in claim 1 or 2 in any of the following: (1) Application in the preparation of temperature-sensitive protein assemblies; (2) Application in the preparation of matrix metalloproteinase-9 responsive protein deassembly and assembly system; (3) Application in the preparation of targeted drug delivery vectors.

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