An injectable self-healing underwater protein and its uses

Abstract

This invention relates to the field of underwater adhesive materials, and particularly to an injectable self-healing underwater protein and its uses. The invention provides a fusion protein comprising a mussel byssal protein fragment and a coiled-helical structure fragment. This invention involves genetic recombination of mussel byssal protein and the coiled-helical structure, thereby providing a novel fusion protein that can self-assemble in solution to form a gel material, possessing both the interfacial adhesiveness of mussel byssal protein and the intrinsic adhesiveness of the coiled-helical structure, thus achieving super-strong underwater adhesion.

Description

Technical Field

[0001] This invention relates to the field of underwater adhesive materials, and in particular to an injectable self-healing underwater protein and its uses. Background Technology

[0002] Underwater adhesives, as a novel type of functional material, have gradually gained widespread attention in recent years and are increasingly needed in various fields. The most typical examples are biomedicine and marine engineering. In biomedicine, traditional treatments use surgical screws and sutures to close wounds. However, these methods often cause considerable pain to patients, and the screws must be removed after healing, often leading to inflammation and infection risks. Therefore, researchers have turned their attention to adhesives, hoping to create new wound treatment methods. Since human tissue exists in a very complex aquatic environment, developing medical materials that maintain their adhesive properties in high humidity or aquatic environments is of great significance. In marine engineering, the repair of ships, the filling of cracks in subsea oil pipelines and river dams all rely heavily on the development of underwater adhesives, making the demand for high-performance underwater adhesives increasingly urgent.

[0003] Although underwater adhesive materials are needed by society, the preparation of multifunctional underwater adhesive materials with high bonding strength remains a challenging problem. However, some marine organisms have provided researchers with much inspiration. By studying the adhesive proteins secreted by marine or underwater organisms such as mussels, sand worms, caddisflies, and barnacles, the underwater adhesive mechanisms of many adhesive proteins have been gradually revealed, leading to the development of an increasing number of biomimetic underwater adhesive materials. Research in this area includes: 1) Research on biomimetic underwater adhesive materials based on the catechol structure: using chemical methods to modify the catechol structural groups into the main chain of polymers; 2) Research on biomimetic underwater adhesive materials based on condensed composite structures: using molecules with opposite charges, mixing them, and forming spherical particle aggregates under electrostatic and hydrophobic effects to play a role in underwater adhesion; 3) Research on biomimetic underwater adhesive materials based on the amyloid protein structure: using genetic engineering technology to fuse mussel byssal proteins (Mfp-3 and Mfp-5) with the CsgA component of Escherichia coli amyloid protein coiled fiber subunits to form recombinant proteins CsgA-Mfp3 and Mfp5-CsgA, allowing both protein modules to exert adhesive properties simultaneously. Although natural underwater adhesive materials are mainly composed of proteins, most biomimetic underwater adhesive materials at present are prepared based on chemical synthesis methods. The underwater adhesive features injected into these molecules are relatively simple, so the prepared adhesive materials do not have the high adhesive strength of natural adhesive materials in the aquatic environment. Furthermore, poor injectability limits the widespread application of these materials in various fields, especially in the biomedical field. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for preparing a fusion protein and a colloid containing the fusion protein, as well as its uses, to solve the problems in the prior art.

[0005] To achieve the above and other related objectives, the first aspect of the present invention provides a fusion protein comprising a mussel byssal protein fragment and a coiled helical structure fragment.

[0006] In some embodiments of the present invention, the mussel byssal protein is selected from purple mussel byssal protein or Mediterranean mussel byssal protein.

[0007] In some embodiments of the present invention, the mussel byssal protein is selected from mefp-3, mefp-5, Mgfp-3, Mgfp-5, mfp-3s, or mfp3s-pep.

[0008] In some embodiments of the present invention, the mussel byssal protein fragment is: a) a polypeptide fragment with an amino acid sequence as shown in SEQ ID No. 4-9;

[0009] Or b) a polypeptide fragment whose amino acid sequence has more than 80% homology with SEQ ID NO.4-9 and has the function of the polypeptide fragment defined in a).

[0010] In some embodiments of the present invention, the byssal protein is derived from mussels.

[0011] In some embodiments of the present invention, the coiled helical structure fragment is selected from leucine zipper fragments, A1 fragments, ZE / ZR fragments, GCN4 fragments, and γ-ray fragments. 52-88 KI fragment, [(AG)3PEG]10 fragment, PC x P-fragment, NLP fragment.

[0012] In some embodiments of the present invention, the leucine zipper fragment includes an A fragment, an S fragment, and a P fragment;

[0013] The A fragment is: c) a polypeptide fragment with an amino acid sequence as shown in SEQ ID No. 1; or d) a polypeptide fragment with an amino acid sequence having more than 80% homology with SEQ ID No. 1 and having the function of the polypeptide fragment defined in c);

[0014] The S fragment is: e) a polypeptide fragment with an amino acid sequence as shown in SEQ ID No. 2; or f) a polypeptide fragment with an amino acid sequence having more than 80% homology to SEQ ID No. 2 and having the function of the polypeptide fragment defined in e);

[0015] The P fragment is: g) a polypeptide fragment with an amino acid sequence as shown in SEQ ID No. 3; or h) a polypeptide fragment with an amino acid sequence having more than 80% homology with SEQ ID NO. 3 and having the function of the polypeptide fragment defined in g).

[0016] In some embodiments of the present invention, the fusion protein further includes functional protein fragments.

[0017] In some embodiments of the present invention, the leucine zipper fragment includes, from the N-terminus to the C-terminus, an A-terminus, an S-terminus, and a P-terminus.

[0018] In some embodiments of the present invention, the mussel byssal protein fragment is inserted into a leucine zipper fragment.

[0019] In some embodiments of the present invention, the fusion protein comprises, from the N-terminus to the C-terminus, a fragment A, a fragment S, a mussel byssal protein fragment, and a fragment P.

[0020] In some embodiments of the present invention, the fusion protein is modified with tyrosinase.

[0021] In some embodiments of the present invention, the functional protein fragment is selected from one or more combinations of adhesive protein fragments, cell adhesion peptide fragments, enzyme protein fragments, growth factor fragments, antibacterial active peptide fragments, coagulation factor fragments, biocompatible peptide fragments, antibiotic peptide fragments, and monitoring peptide fragments.

[0022] In some embodiments of the present invention, the fusion protein comprises, from the N-terminus to the C-terminus, a fragment A, a fragment S, a functional protein fragment, a mussel byssal protein fragment, and a fragment P.

[0023] A second aspect of the present invention provides an isolated polynucleotide encoding a fusion protein as described above.

[0024] A third aspect of the present invention provides a construct containing the isolated polynucleotides as described above.

[0025] A fourth aspect of the present invention provides an expression system comprising an exogenous polynucleotide as described above integrated into a construct or genome as described above.

[0026] The fifth aspect of the present invention provides a method for preparing the fusion protein, comprising: culturing the expression system as described above under conditions suitable for expressing the fusion protein.

[0027] In some embodiments of the present invention, the expressed fusion protein is modified with tyrosinase.

[0028] A sixth aspect of the present invention provides a self-assembling colloidal solution comprising the fusion protein described above.

[0029] A seventh aspect of the present invention provides a colloid comprising the fusion protein described above.

[0030] In some embodiments of the present invention, the colloid is a hydrogel.

[0031] In some embodiments of the present invention, the content of the fusion protein in the colloid is 70 mg / mL to 250 mg / mL.

[0032] In some embodiments of the present invention, the colloid is obtained by the self-assembly of the fusion protein.

[0033] In some embodiments of the present invention, the colloid is prepared from the self-assembled colloidal solution of claim 9.

[0034] In some embodiments of the present invention, the mussel byssal protein in the colloid is selected from a combination of mefp-3 and mefp-5.

[0035] The eighth aspect of the present invention provides the use of the fusion protein, the self-assembled colloidal solution, and the colloidal substance in the field of underwater adhesive preparation. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of a modular construction method for underwater adhesive materials.

[0037] Figure 2 A schematic diagram illustrating the bonding principle of underwater adhesive materials.

[0038] Figure 3 The constructed recombinant ASP plasmid map.

[0039] Figure 4 Map of the recombinant AS-Mefp3-P plasmid.

[0040] Figure 5 Map of the recombinant AS-Mefp5-P plasmid.

[0041] Figure 6 Map of the constructed recombinant AS-Spytag-Mefp3-P plasmid.

[0042] Figure 7 Map of the constructed recombinant AS-Snooptag-Mefp5-P plasmid.

[0043] Figure 8To verify the successful expression and purification of AS-Mefp3-P, AS-Mefp5-P, and ASP using Coomassie Brilliant Blue staining and Western blotting assays. (a) Coomassie Brilliant Blue staining (left) and Western blotting identification (right) of AS-Mefp3-P and AS-Mefp5-P; (b) standard protein markers; (c) Coomassie Brilliant Blue staining (left) and Western blotting identification (right) of ASP.

[0044] Figure 9(a) shows the circular dichroism spectrum in the circular dichroism experiment.

[0045] Figure 9(b) shows the percentage of secondary structures in the circular dichroism chromatogram experiment.

[0046] Figure 10 The dopa structure of the modified protein was verified by staining with nitrotetrazole blue. In the figure, AS3P and AS5P represent unmodified AS-Mefp3-P and AS-Mefp5-P protein samples, respectively, while AS3P MO and AS5P MO represent tyrosinase-modified AS-Mefp3-P and AS-Mefp5-P protein samples, respectively.

[0047] Figure 11 The morphology of the ASP series proteins was characterized using AFM. (a) and (b) show the morphology of AS-Mefp3-P protein before and after tyrosinase modification, respectively; (c) and (d) show the morphology of AS-Mefp5-P protein before and after tyrosinase modification, respectively; (e) and (f) show the morphology of a mixture of equal mass ratios of AS-Mefp3-P and AS-Mefp5-P protein before and after tyrosinase modification, respectively; and (g) shows the morphology of the ASP protein.

[0048] Figure 12 This is a hydrogel diagram of ASP series proteins. In the diagram, ASP refers to hydrogels formed by ASP proteins; A3 and A3 MO refer to hydrogels formed by unmodified and modified AS-Mefp3-P proteins, respectively; A5 and A5 MO refer to hydrogels formed by unmodified and modified AS-Mefp5-P proteins, respectively; and A35 and A35 MO refer to hydrogels formed by an equal mass mixture of unmodified and modified AS-Mefp3-P and AS-Mefp5-P proteins, respectively.

[0049] Figure 13 (a) shows the adhesive strength of the ASP series lap shear test, and (b) shows the lap shear test environment.

[0050] Figure 14Underwater adhesion lap shear test for A35 MO and ASP: (a) shows the adhesion strength of the ASP series; (b) shows the lap shear test environment.

[0051] Figure 15 For bone adhesion test.

[0052] Figure 16 This is a comparison chart of the adhesion test results for A35 MO10 and A35 MO. The yellow bars represent an air humidity environment of 30%, and the purple bars represent a water environment.

[0053] Figure 17 SEM images of ASP and A35 MO after freeze-drying. (a) and (b) are cross-sectional SEM images of ASP, and (c) and (d) are cross-sectional SEM images of A35 MO. The scale bar for (a) and (c) is 20 μm, and the scale bar for (b) and (d) is 10 μm.

[0054] Figure 18 Amplitude scanning tests were performed on ASP and A35 MO.

[0055] Figure 19 Frequency sweep test for ASP and A35 MO. Red represents A35 MO, black represents ASP; solid dots represent G', hollow dots represent G.

[0056] Figure 20 This experiment investigates the interaction between AS-Mefp3-P and AS-Mefp5-P. (a) ASpy, (b) ASnoop, (c) Aspy and Asnoop brought close together, (d) Interface state under a fluorescence microscope, and (e) Interface state after 10 minutes.

[0057] Figure 21 This is an experiment to investigate the self-healing properties of A35 MO. (a) is a complete A35 MO. (b) is the shape after the hydrogel is cut in half from the middle with a blade. (c) is the shape after the two parts of the hydrogel are separated. (d) is the shape after the two parts of the hydrogel are brought close together and fused. (e) is the state of the fused hydrogel being lifted up.

[0058] Figure 22 This study explores the application of A35 MO's self-healing capabilities in the repair of porous polyurethane materials.

[0059] Figure 23 Demonstration of the injectable A35 MO (a) and a 3D printed pattern (b).

[0060] Figure 24 A material for filling missing glass microspheres by utilizing the injectable properties of A35 MO.

[0061] Figure 25 This is a specific application example of A35 MO underwater bonding. Detailed Implementation

[0062] Through extensive exploratory experiments, the inventors of this invention developed a powerful underwater adhesive protein material with multiple functions (injectability and self-healing properties) using a gene modularization method. This novel multifunctional adhesive material has broad application prospects in multiple fields, especially in the biomedical field, and this invention was completed based on this.

[0063] In one aspect, the present invention provides a fusion protein, which may include mussel byssal protein fragments and leucine zipper fragments.

[0064] The fusion protein provided by this invention may include mussel foot proteins. These mussel foot proteins may be selected from, but are not limited to, mytilus edulis foot protein (mefp) and Mediterranean mussel foot protein (Mytilus galloprovincialis foot protein), and more specifically from, but are not limited to, mefp-3, mefp-5, Mgfp-3, Mgfp-5, mfp-3s, mfp3s-pep, or variants thereof. For example, the mussel foot protein fragment may be: a) a polypeptide fragment with an amino acid sequence as shown in one of SEQ ID No. 4-9, or b) a polypeptide fragment with an amino acid sequence having 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more homology to one of SEQ ID No. 4-9, and having the function of the polypeptide fragment defined in a). The amino acid sequence in b) specifically refers to, as shown in SEQ ID No. The amino acid sequence shown in any one of SEQ ID No. 4-9 is obtained by substitution, deletion, or addition of one or more amino acids (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3), or by adding one or more amino acids (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) to the N-terminus and / or C-terminus, and the polypeptide fragment it encodes has the function of the polypeptide fragment encoded by the amino acid sequence shown in any one of SEQ ID No. 4-9. The mussel byssal protein is typically derived from mussels (Latin name: Mytilidae), and more specifically from the purple mussel (Mytilus eduli), Mediterranean mussel (Mytilus galloprovinciali), etc.

[0065] The fusion protein provided by this invention may include coiled-coiled fragments, which typically enable the fusion protein to self-assemble into an α-helix conformation. These coiled-coiled fragments may be leucine zipper fragments, A1 fragments (e.g., polypeptide fragments encoded by the amino acid sequences shown in SGDLXNXVAQLXRX (SEQ ID No. 10), VRSLXDXAAELXQX (SEQ ID No. 11), VSRLXNXIEDLXAXI (SEQ ID No. 12), where X is E or K), ZE / ZR fragments, GCN4 fragments, γ... 52-88 KI fragments (e.g., peptide fragments encoded by the amino acid sequence shown in SEQ ID No. 13), PC xP fragment, NLP (Nucleoporin-like polypeptide) fragment, etc. In a specific embodiment of the present invention, the coiled-helical structure fragment can be a leucine zipper fragment, which typically includes an A fragment, an S fragment, and a P fragment. The A fragment can be: c) a polypeptide fragment with an amino acid sequence as shown in SEQ ID No. 1, or d) a polypeptide fragment with an amino acid sequence having 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more homology to SEQ ID No. 1, and having the function of the polypeptide fragment defined in c). The S fragment can be: e) a polypeptide fragment with an amino acid sequence as shown in SEQ ID No. 2, or f) a polypeptide fragment with an amino acid sequence having 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more homology to SEQ ID No. 2, and having the function of the polypeptide fragment defined in e). The P fragment can be: g) a polypeptide fragment with an amino acid sequence as shown in SEQ ID No. 3, or h) a polypeptide fragment with an amino acid sequence having 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more homology to SEQ ID No. 3 and having the function of the polypeptide fragment defined in g). The amino acid sequences in d), f), and h) specifically refer to: amino acid sequences obtained by substituting, deleting, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively; or amino acid sequences obtained by adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminus and / or C-terminus, respectively, and whose encoded polypeptide fragments have the function of the polypeptide fragments encoded by the amino acid sequences shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3. In a specific embodiment of the present invention, the leucine zipper fragment includes, from the N-terminus to the C-terminus, an A fragment, an S fragment, and a P fragment.

[0066] In the fusion protein provided by this invention, the mussel byssal protein fragment can be inserted into the leucine zipper fragment. In a specific embodiment of this invention, the fusion protein comprises, from the N-terminus to the C-terminus, fragments A, S, mussel byssal protein, and P.

[0067] The fusion protein provided by this invention can be modified with tyrosinase. Tyrosinase modification typically refers to the conversion of at least a portion of the tyrosine groups in the fusion protein into dopa groups (DOPA groups, 3,4-dihydroxyphenylalanine). Generally, the conversion rate of the tyrosine groups can be 30%–80%, 50%–65%, 30%–35%, 35%–40%, 40%–45%, 45%–50%, 50%–55%, 55%–60%, 60%–65%, 65%–70%, 70%–75%, or 75%–80%. In a specific embodiment of this invention, the tyrosinase used for tyrosinase modification of the fusion protein is the SIGMA-ALDRICH reagent.

[0068] The fusion protein provided by this invention may further include functional protein fragments. These functional protein fragments may be one or more combinations of adhesive protein fragments, cell adhesion peptide fragments, enzyme protein fragments, growth factor fragments, antibacterial active peptide fragments, coagulation factor fragments, biocompatible peptide fragments, antibiotic peptide fragments, and monitoring peptide fragments. The adhesive protein fragments may be, for example, Mfp3s fragments or MFPfast fragments. The cell adhesion peptide fragments may be, for example, RGD fragments (see 2. Heilshorn, SC; Liu, JC; Tirrell, DA, Cell-binding domain context affects cell behavior on engineered proteins. Biomacromolecules 2005, 6(1), 318-323.) or REDV fragments (see Girotti, A.; Reguera, J.; Rodríguezcabello, JC; Arias, FJ; Alonso, M.; Maestera, A., Design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for For tissue engineering purposes. Journal of Materials Science Materials in Medicine 2004, 15(4), 479-484., etc., the enzyme protein fragment (e.g., toxic substance degrading enzyme) can be, for example, AdhD fragment (see Wheeldon, IR; Campbell, E.; Banta, S., A Chimeric Fusion Protein Engineered with Disparate Functionalities—Enzymatic Activity and Self–assembly. Journal of Molecular Biology 2009, 392(1), 129-142.), OPH fragment (see Girotti, A.; Reguera, J.; Rodríguezcabello, JC; Arias, FJ; Alonso, M.; Maestera, A.), etc., the enzyme protein fragment (e.g., toxic substance degrading enzyme) can be, for example, AdhD fragment (see Wheeldon, IR; Campbell, E.; Banta, S., A Chimeric Fusion Protein Engineered with Disparate Functionalities—Enzymatic Activity and Self–assembly. Journal of Molecular Biology 2009, 392(1), 129-142.), OPH fragment (see Girotti, A.; Reguera, J.; Rodríguezcabello, JC; Arias, FJ; Alonso, M.; Maestera, A.), etc.Design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes. Journal of Materials Science Materials in Medicine 2004, 15(4), 479-484., etc., the growth factor fragments may be, for example, VEGF fragments (see Zisch, AH; Schenk, U.; Schense, JC; Sakiyama-Elbert, SE; Hubbell, JA, Covalently conjugated VEGF–fibrin matrices for endothelialization. Journal of Controlled Release 2001, 72(1), 101-113.), KGF fragments (see Koria, P.; Yagi, H.; Kitagawa, Y.; Megeed, Z.; Nahmias, Y.; Sheridan, R.; Yamush, ML, Self-assembling elastin-like peptides growth factor chimeric nanoparticles for the treatment of chronic The antimicrobial active polypeptide fragments may be, for example, HNP-2,4 fragments (see Gomes, SC; Leonor, IB; Mano, JF; Rui, LR; Kaplan, DL, Antimicrobial functionalized genetically engineered spider silk. Biomaterials 2011, 32(18), 4255-4266.) or Hepcidin fragments (see Gomes, SC; Leonor, IB; Mano, JF; Rui, LR; Kaplan, DL, Antimicrobial functionalized genetically engineered spider silk. Biomaterials 2011, 32(18), 4255-4266.), Antimicrobial functionalized genetically engineered spider silk. Biomaterials2011, 32(18), 4255-4266.), Indolicidin fragments (see Tsai, CW; Hsu, NY; Wang, CH; Lu, CY; Chang, Y.; Tsai, HH; Ruaan, RC, Coupling molecular dynamics simulations with experiments for the rational design of indolicidin-analogousantimicrobial peptides.Journal of Molecular Biology 2009,392(3),837-854.), Plectasin fragment (see Zakeri, B.; Lu, TK, Synthetic Biology of AntimicrobialDiscovery.Acs Synthetic Biology 2013, 2(7), 358-372.), β-defensin-1 fragment (see Zakeri, B.; Lu, TK, Synthetic Biology of Antimicrobial Discovery.Acs Synthetic Biology 2013, 2(7), 358-372., etc., the coagulation factor fragments may be, for example, coagulation factor III fragments (see Edgington, TS; Mackman, N.; Brand, K.; Ruf, W., The structural biology of expression and function of tissue factor. Thrombosis & Haemostasis 1991, 66(1), 67.), coagulation factor V fragments (see Dahlback, B.; Hildebrand, B., Inherited Resistance to Activated Protein C is Corrected by Anticoagulant Cofactor Activity Found to be a Property of Factor V.Proceedings of the National Academy of Sciences of the United States of America 1994, 91(4), 1396-1400.), coagulation factor VII fragment (see O'Hara, PJ; Grant, FJ; Haldeman, BA; Gray, CL; Insley, MY; Hagen, FS; Murray, MJ, Nucleotide sequence of the gene coding for human factor VII, a vitamin K-dependent protein participating in blood coagulation. Proceedings of the National Academy of Sciences of the United States of America 1987, 84(15), 5158-5162.), etc., the biocompatible polypeptide fragments generally refer to polypeptide fragments that enhance the biocompatibility of fusion proteins, the antibacterial peptide fragments generally refer to polypeptide fragments that inhibit microbial growth and / or kill microorganisms, and the monitoring polypeptide fragments can be, for example, spytag, snooptag, etc. In one specific embodiment of the present invention, the functional protein fragment is a spytag and / or a snooptag, and the amino acid sequence of the functional protein fragment is shown in SEQ ID No. 14 and SEQ ID No. 15, thereby enabling it to react with a fusion protein fused with Spycatcher- (amino acid sequence shown in SEQ ID No. 16) and Snoopcatcher (amino acid sequence shown in SEQ ID No. 17). In another specific embodiment of the present invention, the fusion protein comprises, from the N-terminus to the C-terminus, a fragment A, a fragment S, a functional protein fragment, a mussel byssal protein fragment, and a fragment P.

[0069] Another aspect of the present invention provides an isolated polynucleotide encoding a fusion protein as described above.

[0070] In another aspect, the present invention provides a construct containing the isolated polynucleotides as described above. The method for constructing the construct using the isolated polynucleotides as described above should be known to those skilled in the art. For example, the construct can be constructed by inserting the isolated polynucleotides into the multiple cloning site of an expression vector. Those skilled in the art can select a suitable expression vector for constructing the construct. For example, the expression vector can be a bacterial plasmid or similar vector; another example is an E. coli plasmid; in a specific embodiment of the present invention, the expression vector can be a pHis-FUS plasmid or similar.

[0071] Another aspect of the present invention provides an expression system containing exogenous polynucleotides as described above integrated into the construct or genome as described above. The expression system is typically a host cell; any cell suitable for expression of the construct can serve as a host cell. For example, the host cell can be a prokaryotic cell, or, for another example, a bacterial cell. In one specific embodiment of the present invention, the host cell can be, for example, Escherichia coli cells.

[0072] Another aspect of the present invention provides a method for preparing the fusion protein, comprising: culturing the expression system as described above under conditions suitable for expressing the fusion protein. Those skilled in the art can select appropriate culture conditions based on the type of host cells used. For example, the culture medium used in the culture can be various conventional culture media, and those skilled in the art can select an appropriate culture medium based on experience, and culture under conditions suitable for host cell growth. For another example, after the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature change or chemical induction), and the cells are cultured for a further period. Generally, the fusion protein described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified using its physical, chemical, and other properties through various separation methods. These methods should be known to those skilled in the art; for example, methods such as refolding, treatment with a protein precipitant (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and other various liquid chromatography techniques can be used.

[0073] The method for preparing the fusion protein provided by the present invention may further include: modifying the expressed fusion protein with a tyrosinase. Methods for modifying compounds (e.g., the fusion protein as described above) with tyrosine should be known to those skilled in the art; for example, the fusion protein may be placed in the presence of a tyrosinase.

[0074] Another aspect of the present invention provides a self-assembling colloidal solution comprising the fusion protein as described above. The self-assembling colloidal solution is typically in solution form (e.g., aqueous solution) and can form a colloid under suitable conditions. Those skilled in the art can select a suitable type and amount of solvent to mix with the fusion protein to form the solution of the self-assembling colloid; for example, it can be a PBS solution (e.g., PBS buffer provided by Shanghai ABCONE Biotechnology Co.). Furthermore, those skilled in the art can appropriately adjust the concentration of the fusion protein in the solution according to the content of the fusion protein in the prepared colloid.

[0075] In another aspect, the present invention provides a colloid comprising the fusion protein as described above. The colloid is typically a hydrogel, i.e., a gel with water as the dispersion medium. The colloid can be prepared from a self-assembled colloidal solution as described above, primarily utilizing the self-assembly of the fusion protein therein to obtain the colloid. In the prepared colloid, the content of the fusion protein is typically between 70 mg / mL and 250 mg / mL.

[0076] The self-assembled colloidal solution and / or colloid provided by this invention can be single-component, i.e., containing only one fusion protein as described above, or multi-component, i.e. containing two or more fusion proteins as described above. In a specific embodiment of this invention, the fusion protein contained in the self-assembled colloidal solution and / or colloid may be a combination of mefp-3 and mefp-5. For example, it may include a fusion protein containing a mefp-3 peptide and a fusion protein containing a mefp-5 peptide, and the ratio of the two may be 1:0.01-100, 1:0.1-10, 1:0.1-0.3, 1:0.3-0.5, 1:0.5-1, 1:1-2, 1:2-3, 1:3-5, or 1:5-10.

[0077] Another aspect of the present invention provides the use of the fusion protein, self-assembled colloidal solution, and colloid as described above in the field of underwater adhesive preparation, and more specifically, a biomimetic underwater adhesive material.

[0078] This invention provides a fusion protein comprising mussel byssal proteins and coiled-coil structural proteins or protein domains. A recombinant plasmid was successfully constructed using molecular biology techniques, transformed into *E. coli*, and the recombinant protein was expressed. Protein expression was identified by polyacrylamide gel electrophoresis and Western blotting, and the effect of the fusion protein on the secondary structure of the recombinant protein was analyzed by circular dichroism spectroscopy. To enhance the interfacial adhesion of the protein, the fusion protein was modified with tyrosinase, and verified by NBT staining. The morphology of the mixed protein under tyrosinase-modified and unmodified conditions was characterized by atomic force microscopy to compare the adsorption capacity of various proteins for mica flakes and their self-aggregation ability. Furthermore, this invention further prepared a hydrogel from the fusion protein. The adhesive force of the hydrogel was tested using a universal testing machine with overlap shear test. The results showed that the maximum adhesive force of the protein hydrogel reached 286 kPa under 30% air humidity and nearly 100 kPa under aqueous conditions, which were 8.8 times and 16 times that of the control protein, respectively. Furthermore, the significantly enhanced adhesive strength of the protein hydrogel was verified through overlap shear tests, scanning electron microscopy, and rheological tests. The overall enhanced adhesive strength of the material stemmed from the simultaneous improvement of its interfacial and intrinsic adhesion. Further, the inventors discovered the changes observed under a fluorescence microscope at the interface between AS-Spytag-Mefp3-P and AS-Snooptag-Mefp5-P after tyrosinase modification, revealing an interaction between AS-Mefp3-P and AS-Mefp5-P within A35 MO. Thus, through self-healing experiments, 3D printing experiments, and underwater adhesion tests on bone and glass slides, the self-healing and injectability of the hydrogel material, as well as the feasibility of underwater adhesion, demonstrate its promising application prospects.

[0079] In summary, this invention recombines mussel byssal proteins with a coiled helical structure to provide a novel fusion protein. This fusion protein can self-assemble in solution to form a gel material, possessing both the interfacial adhesiveness of mussel byssal proteins and the intrinsic adhesiveness of the coiled helical structure, thereby achieving super-strong underwater adhesion. Compared with existing technologies, this invention simultaneously enhances both the interfacial and intrinsic adhesiveness of the material, thereby improving the overall adhesive force, resulting in a multifunctional underwater adhesive material with self-healing and injectability. It explores a new strategy for constructing underwater adhesive materials and demonstrates the potential applications of this material through examples.

[0080] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0081] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0082] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0083] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature; see Sambrook et al., *MOLECULAR CLONING: A LABORATORY MANUAL*, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., *CURRENT PROTOCOLS IN MOLECULAR BIOLOGY*, John Wiley & Sons, New York, 1987 and periodic updates; *theseries METHODS IN ENZYMOLOGY*, Academic Press, San Diego; Wolffe, *CHROMATINSTRUCTURE AND FUNCTION*, Third edition, Academic Press, San Diego, 1998; *METHODS IN ENZYMOLOGY*, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and *METHODS IN MOLECULAR*. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0084] In this embodiment of the invention, unless otherwise specified, the enzyme ligation reaction system (10 μL) is as shown in Table 1:

[0085] Table 1

[0086]

[0087] In this embodiment of the invention, unless otherwise specified, the reaction system (50 μL) used for PCR amplification and PCR identification comprises the following components:

[0088]

[0089] The PCR reaction conditions were: 98℃ pre-denaturation for 20s, 98℃ for 10s, 58℃ for 30s, 72℃ for 45s, 35 cycles, and a final extension at 72℃ for 5min, with deionized water as a negative control.

[0090] In this embodiment of the invention, unless otherwise specified, the LB culture medium used was purchased from (Taiwan Sangon Biotech, L001-1kg), and the antibiotic solution was prepared as follows: carbenicillin (macklin, C805408) was prepared into an aqueous solution of 100mg / ml and stored at -20℃. The final concentration of the antibiotic was 50ng / ml when used.

[0091] In this embodiment of the invention, unless otherwise specified, the formulations of the lysis buffer, washing solution, eluent, and dialysis solution used are as follows:

[0092] (1) Liquefaction buffer: 8M guanidine hydrochloride aqueous solution

[0093] (2) Cleaning solution: 20mM imidazole aqueous solution

[0094] (3) Eluent: 1000mM imidazole aqueous solution

[0095] (4) Dialysate: dd H2O

[0096] The reagents and consumables used in the embodiments of this invention are shown in Table 2:

[0097] Table 2

[0098]

[0099] The Escherichia coli BL21(DE3) used in the embodiments of this invention can be purchased from TransGene Biotech (Beijing), catalog number CD601.

[0100] The pHis-FUS(LC) plasmid referenced in the embodiments of this invention (Cell-free Formation of RNAGranules: Low Complexity Sequence Domains Form Dynamic Fibers within Hydrogels. 2012, Cell, Volume 149, Issue 4, 11 May 2012, Pages 753–767. Masato Kato, Tina W. Han, Steven L. McKnight) has been provided to the applicant by Professor McKnight's research group at Southwestern Medical College, USA.

[0101] Example 1

[0102] Construction, expression, and purification of functional ASP fusion proteins:

[0103] The ASP recombinant plasmid was constructed based on the existing pHis-FUS(LC) plasmid in the laboratory, and the target gene fragment was inserted. The amino acid sequence of A is shown in SEQ ID NO:1, the amino acid sequence of S is shown in SEQ ID NO:2, the amino acid sequence of P is shown in SEQ ID NO:3, and the amino acid sequence of ASP is shown in SEQ ID NO:20. The main steps involved in constructing the above-mentioned expression plasmid include:

[0104] (1) Insert the S fragment into the vector pHis-FUS(LC): The S protein gene fragment was synthesized by Suzhou Genewise Technology Co., Ltd. and inserted into the pHis-FUS(LC) plasmid to obtain the vector pHis-S-FUS(LC). The restriction sites at both ends of S are NcoI and BamHI.

[0105] (2) Insertion of fragment A into vector pHis-S-FUS(LC): Gene fragment A was amplified using primers (template not required). The DNA sequence of the forward primer AF is shown in SEQ ID NO:21, and the DNA sequence of the reverse primer AR is shown in SEQ ID NO:22. Vector pHis-S-FUS(LC) and gene fragment A obtained by PCR were digested with restriction endonucleases NdeI and NcoI, respectively, at 37℃ for 1 h. After digestion, the digestion products were recovered by gel extraction using the EasyPure Quick Gel Extraction Kit manufactured by Beijing TransGen Biotech Co., Ltd. The recovered products were ligated with T4 ligase at 25℃ for 1 h to obtain vector AS-FUS(LC).

[0106] (3) Inserting the P fragment into the vector AS-FUS(LC): The gene fragment P was amplified using primers (no template required). The DNA sequences of the forward primer PF and the reverse primer PR are detailed in the appendix. The vector AS-FUS(LC) and the PCR-obtained gene fragment P were digested with restriction endonucleases BamHI and XhoI, respectively. The recovered products were ligated using T4 ligase to obtain the target vector ASP. The plasmid map is shown in [link to plasmid map]. Figure 3 .

[0107] Example 2

[0108] Construction, expression, and purification of functional ASMP proteins:

[0109] A recombinant ASMP plasmid was constructed, where M represents peptides with different functional groups (M represents Mefp3, Mefp5, Spytag-Mefp3, and Snooptag-Mefp5, respectively). The ASMP expression plasmid was transformed into BL21(DE3) *E. coli* to express the ASMP fusion protein. The amino acid sequences of Mefp3, Mefp5, Spytag, and Snooptag are shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:14, SEQ ID NO:15, AS-Mefp3-P, AS-Mefp5-P, AS-Spytag-Mefp3-P, and AS-Snooptag-Mefp5-P are shown in SEQ ID NO:32.

[0110] Based on the successfully constructed ASP plasmid, the target gene fragment is inserted. The main steps involved in constructing the required expression plasmid include:

[0111] (1) For the construction of expression plasmids AS-Mefp3-P, AS-Mefp5-P, AS-Spytag-Mefp3-P and AS-Snooptag-Mefp5-P, the expression plasmid ASP was linearized by digesting it with restriction endonucleases HindIII and BamHI according to conventional methods. Based on the gene sequences of Mefp3, Mefp5, Spytag-Mefp3, Snooptag-Mefp5 and the digested expression vector, upstream and downstream specific primers suitable for enzyme digestion ligation were designed.

[0112] (2) The Mefp3, Mefp5, Spytag-Mefp3 and Snooptag-Mefp5 fragments used to construct each plasmid were amplified by PCR.

[0113] For constructing plasmid AS-Mefp3-P: the DNA sequence of the forward primer Mefp3-F for the Mefp3 fragment is shown in SEQ ID NO:24, and the DNA sequence of the reverse primer Mefp3-R is shown in SEQ ID NO:25. The template plasmid for Mefp3 is derived from pET11d-CsgA-Mefp3, referenced in Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nature Nanotechnology 2014,9(10),858-866. Zhong,C.;Gurry,T.;Cheng,AA;Downey,J.;Deng,ZT;Stultz,CM;Lu,TK. The constructed plasmid map AS-Mefp3-P is shown in [link to plasmid map]. Figure 4 .

[0114] For constructing plasmid AS-Mefp5-P: the DNA sequence of the forward primer Mefp5-F for the Mefp5 fragment is shown in SEQ ID NO:27, and the DNA sequence of the reverse primer Mefp5-R is shown in SEQ ID NO:28. The template plasmid for Mefp5 was derived from pET11d-Mefp5-CsgA. (See reference: Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nature Nanotechnology 2014, 9(10), 858-866. Zhong, C.; Gurry, T.; Cheng, AA; Downey, J.; Deng, ZT; Stultz, CM; Lu, TK.) The constructed plasmid map AS-Mefp5-P is shown in [link to relevant documentation]. Figure 5 .

[0115] For constructing plasmid AS-Spytag-Mefp3-P: the DNA sequence of the forward primer Spytag-Mefp3-F for the Spytag-Mefp3 fragment is shown in SEQ ID NO:30, and the DNA sequence of the reverse primer Spytag-Mefp3-R is shown in SEQ ID NO:31. The constructed plasmid map AS-Spytag-Mefp3-P is shown in [image missing]. Figure 6 .

[0116] For constructing plasmid AS-Snooptag-Mefp5-P: the DNA sequence of the forward primer Snooptag-Mefp5-F for the Snooptag-Mefp5 fragment is shown in SEQ ID NO:33, and the DNA sequence of the reverse primer Snooptag-Mefp5-R is shown in SEQ ID NO:34. The constructed plasmid map AS-Snooptag-Mefp5-P is shown in [image missing]. Figure 7 .

[0117] The target fragments (Mefp3, Mefp5, Spytag-Mefp3, and Snooptag-Mefp5) obtained by PCR amplification were digested with restriction endonucleases HindIII and BamHI using conventional enzyme digestion methods. These fragments were then ligated with the linearized ASP vectors obtained from HindIII and BamHI digestion using the T4 enzyme ligation method to obtain the target expression vectors. Based on the length and concentration of each fragment, they were added to EP tubes in the required proportions. The reaction system was incubated overnight at 16°C.

[0118] The transformation steps for recombinant plasmids are as follows:

[0119] (1) Transform 10 μL of ligation products of ASP and AS-Mefp3-P, AS-Mefp5-P, AS-Spytag-Mefp3-P, and AS-Snooptag-Mefp5-P into Escherichia coli clone strain DH5α (Kangwei Century CW08085) respectively. The specific steps are as follows:

[0120] a) Take a centrifuge tube containing 100 μL of DH5α competent cells, place it in an ice box to cool and thaw. When it is thawed, add 10 μL of ligation product, gently mix, and place it in an ice box for 30 min.

[0121] b) Heat shock at 42℃ for 45 seconds, then quickly transfer the centrifuge tube to an ice box and let it stand for 2 minutes.

[0122] c) Add 500 μL of sterile LB medium to the centrifuge tube, mix well, and incubate at 37°C on a shaker for 30 min.

[0123] d) Centrifuge the bacterial culture that has been incubated for 30 min in step c at 1000×g for 2 min, remove about 400 μL of supernatant, and pipette the remaining 100 μL of liquid evenly. Drop it onto LB solid medium containing 50 μg / mL carbenicillin, spread the bacteria evenly with a spreader until the bacterial culture is completely absorbed by the plate, and incubate in an inverted incubator at 37℃ for about 12 h.

[0124] (2) Select single clones and inoculate them into LB liquid medium containing 50 μg / mL carbenicillin resistance. Colony PCR is used to identify the presence of the target gene fragment; if a band is amplified, the plasmid transformation is successful. Plasmids are extracted using a plasmid extraction kit (Tiangen DP103) according to the instructions. Sequencing is performed by Suzhou Genewise Co., Ltd. to further identify the recombinant plasmid. If the sequencing results are correct, the recombinant plasmid is transformed into E. coli competent cells BL21(DE3) (TransGene Biotech CD601). Detailed steps are as follows:

[0125] a) Take a centrifuge tube containing 100 μL of Escherichia coli competent cells BL21(DE3) and place it in an ice box.

[0126] b) After the BL21 competent cells have thawed, add about 1 μL of the extracted plasmid, gently mix with a pipette, and place in an ice box for 30 min.

[0127] c) Heat shock at 42℃ for 45 seconds, then immediately transfer the centrifuge tube to an ice box and let it stand for 2 minutes.

[0128] d) Add 500 μL of sterile LB medium (without antibiotics) to the centrifuge tube, mix well, place the centrifuge tube in a 50 mL centrifuge tube, and incubate at 37°C with shaking for 30 min.

[0129] e) Centrifuge the bacterial culture that has been incubated for 30 min in step d at 1000×g for 2 min, remove about 400 μL of supernatant, and pipette the remaining 100 μL of liquid evenly. Drop it onto LB solid medium containing 50 μg / mL carbenicillin, spread the bacteria evenly with a spreader until the bacterial culture is completely absorbed by the plate, and incubate upside down in a 37℃ incubator for about 12 h.

[0130] The steps for inducing expression and purifying recombinant ASMP protein are as follows:

[0131] (1) Pick BL21(DE3) Escherichia coli containing the target plasmids (ASP, AS-Mefp3-P, AS-Mefp5-P, AS-Spytag-Mefp3-P and AS-Snooptag-Mefp5-P) and inoculate them into 100 mL of LB liquid medium containing 50 μg / mL carbenicillin resistance. Incubate overnight (about 12 h) at 37 °C with shaking on a shaker at 220 rpm.

[0132] (2) Take 10 mL of the overnight culture and inoculate it into 1 L of LB liquid medium containing 50 μg / mL carbenicillin at a ratio of 1:100. Incubate at 37°C with shaking until OD. 600 Between 0.8 and 1.0.

[0133] (3) Add 1 mL of 0.5 M IPTG to the bacterial culture in (2) and induce incubation at 37°C for 3 h by shaking.

[0134] (4) After induction, the bacterial culture is centrifuged to collect the bacterial cells, and the bacterial cells are weighed and stored at -80℃.

[0135] (5) Add 10 mL of 8 M guanidine hydrochloride solution to each gram of bacterial cells and stir for 12 h.

[0136] (6) Centrifuge at 21000×g for 1h to remove bacterial precipitate and retain supernatant. Add Ni-NTA column (General Electric Company, catalog number: 17057501) to supernatant and place on a rotary shaker for 30min.

[0137] (7) Extract protein using a gravity chromatography column (BBI, nickel column). Add the above protein solution to the gravity chromatography column and wash the gravity chromatography column with 200-300 mL of washing solution (20 mM imidazole washing solution) to remove impurities.

[0138] (8) Elute the target proteins (ASP, AS-Mefp3-P, AS-Mefp5-P, AS-Spytag-Mefp3-P and AS-Snooptag-Mefp5-P) bound on the gravity chromatography column with 20-30 mL of elution buffer (1000 mM imidazole washing buffer), collect the flowing target protein solution, and determine the protein concentration using Nanodrop 2000.

[0139] Example 3

[0140] Validation of ASMP fusion protein expression:

[0141] The protein markers were compared and verified using Coomassie brilliant blue staining and Western blotting experiments to check whether the fusion protein could be successfully expressed.

[0142] The main steps of protein polyacrylamide gel electrophoresis are as follows:

[0143] (1) Take 90 μL of the eluted target protein and mix it evenly with 30 μL of 4× loading buffer;

[0144] (2) Add 20 μL of the mixture to the protein gel lane and add standard protein markers as a control;

[0145] (3) Place the protein gel in the electrophoresis buffer and separate it by electrophoresis at 165V for 45 minutes;

[0146] (4) After removing the protein gel, stain it with Coomassie Brilliant Blue solution for 1 hour and then decolorize it (decolorization solution: 50% ddH2O, 40% methanol, 10% acetic acid);

[0147] (5) After 1 hour of decolorization, take a picture using a protein gel scanner.

[0148] The main steps of the Western blot assay for protein identification are as follows:

[0149] (1) Same as protein polyacrylamide gel electrophoresis steps (1) to (3);

[0150] (2) Use a protein gel electroplating instrument to transfer protein gel onto nitrocellulose film;

[0151] (3) Immerse the membrane in a solution containing 20% ​​skim milk powder, shake and incubate for 1 hour, then rinse it three times with TBST solution for 10 minutes each time;

[0152] (4) Immerse the membrane in 20 mL of TBST solution containing antigen diluted 5000 times, shake and incubate for 1 h, then wash with TBST solution 3 times, each time for 10 min;

[0153] (5) Immerse the membrane in 20 mL of TBST solution containing 2000 times diluted diantigen solution, shake and incubate for 1 h, then wash with TBST solution 3 times, each time for 10 min;

[0154] (6) Perform a color development operation on the membrane, and then take a picture using a protein gel scanner.

[0155] The results are as follows Figure 8 As shown, Figure 8 As shown in b, the standard protein marker is used as a reference, and it can be seen that... Figure 8 Images a and 8c show that ASP, AS-Mefp3-P, and AS-Mefp5-P proteins exhibited corresponding target bands at 30–40 kDa, 30–40 kDa, and 20–30 kDa, respectively. (In the figures, AS3P, AS5P, and ASP represent AS-Mefp3-P, AS-Mefp5-P, and ASP protein samples, respectively.) The corresponding target bands are slightly larger than the theoretical molecular weight. This may be because the ASP series proteins have more hydrophobic groups, causing them to migrate slightly slower during electrophoresis than typical proteins, thus appearing above the theoretical position, consistent with empirical values. Simultaneously, all expressed proteins have a 6-histidine tag at their C-terminus. Based on the principle of antigen-antibody interaction, the results of the Western blot assay showed the appearance of target bands at the corresponding positions. In summary, the purified protein is the target protein, thus verifying the successful expression and purification of the protein.

[0156] Example 4

[0157] Characterization of ASP and ASMP secondary structures:

[0158] Circular dichroism spectroscopy was used to characterize the secondary structures of ASP, AS-Mefp3-P, and AS-Mefp5-P to examine whether the components Mefp-3 and Mefp-5 of the fusion protein would significantly alter the molecular conformation. The main steps are as follows:

[0159] (1) Dialyze the freshly prepared protein solution multiple times using deionized water.

[0160] (2) Take 100 μL of the target protein solution and perform circular dichroism chromatographic analysis using a circular dichroism chromatogram at a wavelength of 190-250 nm. The experiment was conducted using a Chirascan spectrometer (Applied Photophysics).

[0161] As shown in circular dichroism chromatogram 9, all three protein samples exhibit a negative peak near 208 nm and a negative peak near 223 nm. Both of these peaks are characteristic of α-helix, confirming that the ASP series proteins all contain the secondary structure of α-helix. 3 ; at the same time according to Figure 9b The percentage results of each secondary structure show that the ASP series proteins contain similar proportions of secondary structures, indicating that the Mefp-3 and Mefp-5 proteins fused in this experiment have no significant impact on the secondary structure of ASP. This suggests that, similar to ASP proteins, AS-Mefp3-P and AS-Mefp5-P also contain α-helix secondary structures. Since Mefp-3 and Mefp-5 are random structures, the α-helix mainly originates from the A and P fragments, suggesting that their gelation mechanism is similar to that of ASP, both stemming from their self-assembly capabilities.

[0162] Example 5

[0163] Modification and validation of recombinant protein ASMP tyrosinase:

[0164] By modifying the fusion proteins AS-Mefp3-P, AS-Mefp5-P, AS-Spytag-Mefp3-P, and AS-Snooptag-Mefp5-P with tyrosinase, the tyrosine residues in them are converted into dopa components, in order to enhance the interfacial viscosity of the hydrogel due to the generation of dopa.

[0165] The main steps of tyrosinase modification are as follows:

[0166] (1) The protein extraction process is as described in "Induction and purification steps of recombinant ASMP protein" in Example 2. After the target protein binds to the nickel column, it is deposited on the gravity chromatography column and the impurities are washed with 100-200 mL of 20 mM imidazole buffer.

[0167] (2) Wash the chromatography column after removing contaminating proteins with 20 mL of PBS buffer;

[0168] (3) Add 4 mL of 0.5 mg / mL tyrosinase solution (formulation: 20 mM sodium borate, 100 mM PBS buffer, 100 mM ascorbic acid, pH = 7.0) to the chromatography column and place it on a rotating shaker to react at room temperature for 2 h;

[0169] (4) Add 20 mL of PBS buffer to wash away tyrosinase;

[0170] (5) Add 20-30 mL of elution buffer (400 mM imidazole buffer) to the gravity chromatography column to elute the target proteins (AS-Mefp3-P, AS-Mefp5-P, AS-Spytag-Mefp3-P and AS-Snooptag-Mefp5-P modified with tyrosinase) bound on the gravity chromatography column, and collect the target protein solution flowing down and determine the protein concentration using Nanodrop 2000.

[0171] To verify whether AS-Mefp3-P and AS-Mefp5-P proteins were successfully modified by tyrosinase to convert tyrosine to dihydroxyphenylalanine, NBT staining was performed on the modified protein samples to characterize them. 4 Nitrotetrazolium chloride (NBT) is a characteristic dye used to identify whether tyrosine in proteins has been converted into catechol or quinone structures. It produces a blue compound under the action of a reducing agent. This staining reaction proves the catechol structure, i.e., whether dopa has been successfully catalyzed and introduced. The main steps are as follows:

[0172] (1) The protein solutions modified with tyrosinase and those not modified were transferred onto nitrocellulose films using a spot transfer device;

[0173] (2) Immerse the membrane in 20 mL of freshly prepared 0.6 mg / mL NBT solution (2 M potassium phosphate and glycine buffer, pH = 10.0) and incubate for 45 min in the dark.

[0174] (3) Wash twice with 10 mL of 0.16 M sodium borate solution, immerse in 20 mL of sodium borate solution overnight for decolorization, and then take a picture.

[0175] The results of NBT staining experiment on proteins modified by tyrosinase are as follows: Figure 10As shown, the tyrosinase-modified protein turned blue after reacting with nitrotetrazole blue, proving that some tyrosine residues in the tyrosinase-modified AS-Mefp3-P and AS-Mefp5-P proteins were converted into dopa structures, indicating that the AS-Mefp3-P and AS-Mefp5-P protein samples were successfully modified by tyrosinase; while the unmodified AS-Mefp3-P and AS-Mefp5-P proteins did not show a blue color, verifying that they do not contain dopa structures.

[0176] Example 6

[0177] Atomic force microscopy (AFM) morphology characterization experiments:

[0178] The surface morphology of the obtained target protein solution was characterized using atomic force microscopy (AFM). The main steps are as follows:

[0179] (1) Add the freshly prepared target protein solution (4 mg / mL) with a concentration of about 1 μM to a clean and flat mica sheet surface, incubate for 12 h, rinse the mica sheet surface with deionized water and dry it with nitrogen gas.

[0180] (2) Morphological scanning was performed using an atomic force microscope in tapping mode. The microprobe used was an AC 160 (k = 26.1 N / m, ν ~ 300 kHz). The experiment was conducted using an Asylum MFP-3D AFM (Asylum Research) instrument.

[0181] The obtained image is as follows Figure 11The results show that, overall, the ASP series proteins all formed protein aggregates without exhibiting any unique morphology. Among all protein solution samples of equal mass, the ASP protein aggregates were smaller in size and had lower adsorption on the mica surface. Comparisons of AS-Mefp3-P, AS-Mefp5-P, and equal masses of AS-Mefp5-P and AS-Mefp3-P solutions, as well as their respective tyrosine-modified solutions, revealed that the tyrosinase-modified protein aggregates were more compact, and their adsorption on the mica surface was higher than that of the unmodified protein samples. It can be inferred that adding the sticky proteins Mefp-3 and Mefp-5 to the leucine zipper domain does not change the morphology of the original leucine zipper protein, while enhancing its adsorption capacity on the mica surface; while the tyrosinase-modified samples showed enhanced protein aggregation and further enhanced adsorption on the mica surface, which may be due to the sticky characteristics of DOPA. Meanwhile, it was found that the most significantly enhanced aggregation ability was observed in the equal-mass AS-Mefp5-P and AS-Mefp3-P modified with tyrosinase. This may be due to the increased intrinsic protein viscosity resulting from the interaction between the modified Mefp-5 and Mefp-3.

[0182] Example 7

[0183] ASP series protein gelation experiments:

[0184] By using a method of lyophilization followed by the addition of PBS solution, ASP series proteins can self-assemble into hydrogels without the addition of chemical cross-linking agents. The main steps are as follows:

[0185] (1) Place the purified protein solution sample in a dialysis bag and dialyze it in deionized water 3 times, each time for 30 minutes.

[0186] (2) The dialysis sample was placed into a 50 mL centrifuge tube and frozen in a -80 °C freezer;

[0187] (3) Take out the completely frozen sample, open the centrifuge tube cap, seal it with sealing film, and make an opening in the film with a needle to ensure gas flow;

[0188] (4) Place the centrifuge tubes in a freeze dryer for 24 hours;

[0189] (5) Take out the sample, weigh it, calculate the volume of the sample at the specified concentration, and add 100mM PBS solution to the required volume;

[0190] (6) Allowing the sample to stand for 3 hours will form a self-assembled hydrogel of a specific concentration.

[0191] like Figure 12The images shown are of the ASP series proteins in their hydrogel state. The ASP protein hydrogel (ASP for short) is colorless and transparent. Unmodified AS-Mefp3-P, AS-Mefp5-P, and equal masses of AS-Mefp3-P and AS-Mefp5-P mixed proteins (referred to as A3, A5, and A35, respectively) are slightly yellowish. The tyrosinase-modified AS-Mefp3-P, AS-Mefp5-P, and equal masses of AS-Mefp3-P and AS-Mefp5-P mixed proteins (referred to as A3MO, A5MO, and A35MO, respectively) are slightly brownish, possibly due to the oxidation of some dopa to quinone. From the gel morphology, the ASP series are generally similar. However, their properties, such as viscoelasticity and viscous strength, require further analysis using rheometer and universal testing machine results.

[0192] Example 8

[0193] Adhesive strength test of ASP series protein adhesives:

[0194] The main steps for testing viscosity using a universal testing machine are as follows:

[0195] (1) Weigh 3 mg of 250 mg / mL hydrogel sample, spread it on a stainless steel sheet (size 10 mm × 50 mm × 0.025 mm, spreading range 10 mm × 10 mm), and adhere the same stainless steel sheet to the spread part.

[0196] (2) Place the sample in a 37°C incubator for 2 hours.

[0197] (3) Take a sample and test it using a universal testing machine at room temperature and 30% humidity. Set the speed to 5 mm / min and the distance between the two clamps to 60 mm.

[0198] (4) For samples tested in water, after sampling, immerse the samples in water at room temperature for 5 minutes, and then test the samples in a water environment using a universal testing machine. Other instrument settings are the same as in (3).

[0199] like Figure 13 As shown, the adhesion strength of A3 and A5 after adding the adhesive proteins Mefp-3 and Mefp-5 through gene modular construction was significantly improved compared to ASP, being 2 times and 3.1 times that of ASP, respectively. The adhesion strength of A3 MO and A5 MO was 2 times and 1.7 times that of the corresponding unmodified hydrogels, respectively, and 4.1 times and 5.2 times that of ASP, respectively. The adhesion strength of A35 MO reached as high as 286.14±24.37 kPa, which was 1.7 times that of the corresponding unmodified hydrogel and 8.8 times that of ASP. Analysis of the above experimental data leads to the following conclusions:

[0200] (1) The adhesive strength of A3 and A5 after adding viscous protein was improved compared with ASP. This is because viscous protein plays a role in enhancing interfacial adhesion.

[0201] (2) Compared to ASP, A3 MO and A5 MO, which were modified with tyrosinase, showed a significant increase in adhesive strength. This is because the dopa component formed after tyrosinase modification further enhances interfacial adhesion through hydrogen bonding and metal coordination. The higher adhesive strength of A5 MO after tyrosinase modification compared to A3 MO is likely due to the presence of more tyrosine and lysine in Mefp-5, resulting in stronger interfacial adhesion. These results are consistent with previous studies indicating that Mfp-5 has stronger adhesive ability than Mfp-3.

[0202] (3) The adhesive strength of A35 MO modified with tyrosinase showed the greatest improvement compared to ASP. Based on the adhesive strength data of A3MO and A5 MO, A5 MO has a higher adhesive strength than A3 MO, suggesting that the adhesive strength of the sample prepared by mixing the two should be between the two. However, the actual adhesive strength obtained was not simply the average of the two, but exceeded that of either sample alone. Therefore, it can be inferred that the significant improvement in the adhesive strength of A35 MO may be due to two factors: First, the dopa component in the tyrosinase-modified AS-Mefp3-P and AS-Mefp5-P proteins promotes the improvement of interfacial adhesion. Stainless steel sheets contain various metals and their oxides, among which metals can form coordinate bonds with dopa, and oxygen in metal oxides can form hydrogen bonds with dopa, thereby increasing interfacial adhesion. Second, the interactions between AS-Mefp3-P and AS-Mefp5-P (derived from molecular recognition of the leucine zipper and intermolecular hydrogen bonding between Mefp-3 and Mefp-5) may make their crosslinking network more compact, leading to increased intrinsic viscosity. Since the interfacial viscosity and intrinsic viscosity of A35 MO are simultaneously enhanced compared to ASP, these two factors jointly improve the adhesive strength of the hydrogel.

[0203] This experiment successfully demonstrated that the hydrogel formed by an equal mass mixture of tyrosinase-modified AS-Mefp3-P and AS-Mefp5-P proteins exhibits the highest overall adhesive strength, exceeding that of individual AS-Mefp3-P and AS-Mefp5-P protein hydrogels under the same conditions, regardless of whether the proteins were modified or not. Furthermore, in Lu's research on underwater adhesive materials, experiments using AFM mechanical testing showed... 2Two types of self-assembling adhesive nanofibers, CsgA-Mfp3 and Mfp5-CsgA, obtained by fusing expression of E. coli amyloid protein CsgA and mussel byssal proteins Mfp-3 and Mfp-5, achieved maximum adhesiveness when mixed in an equimolar ratio, both exceeding the performance of CsgA-Mfp3 and Mfp5-CsgA. This demonstrates that regardless of whether macroscopic (overlap shear test) or microscopic (AFM) experimental methods are used, the adhesiveness of the mixed adhesive fusion proteins containing Mfp-3 and Mfp-5 is significantly enhanced, exceeding the effect of each individual adhesive fusion protein. Therefore, it can be inferred that the fusion protein molecules containing Mfp-3 and Mfp-5 interact after mixing, strengthening the adhesive properties; this characteristic is exhibited not only at the microscopic level but also at the macroscopic level.

[0204] like Figure 14 As shown, the adhesive forces of ASP and A35 MO hydrogels decreased by approximately 5.3 times and 2.9 times, respectively, compared to the adhesive forces measured in air under the same incubation conditions. However, A35 MO still exhibited a higher adhesive force, reaching nearly 100 kPa, which is 16 times that of ASP underwater. Furthermore, it is evident that the decrease in adhesive force of A35 MO underwater compared to its air-measured adhesive force was less than that of ASP, indicating that A35 MO demonstrates superior performance in underwater adhesion compared to ASP.

[0205] To verify the principle behind the significant improvement in the adhesive strength of A35 MO, this invention conducted an overlap shear test on the hydrogel of A35 MO after it had been left to stand for ten days (hereinafter referred to as A35 MO10). The experimental results are as follows: Figure 16 As shown, the adhesive strength of A35 MO10 measured in air at 21°C and 30% humidity was 80.94 kPa, only 29.16% of that of A35 MO. In the corresponding underwater test, the adhesive strength was 23.64 kPa, only 24.22% of that of A35 MO. From these data, it can be inferred that the dopa structure in A35 MO, after tyrosinase modification, can be oxidized to a quinone structure in air, significantly reducing its interfacial viscosity. Therefore, compared to A35 MO, the adhesive strength of A35 MO10 is significantly lower in both air and water environments. However, it can also be observed that the adhesive strength of A35 MO10 is still stronger than that of ASP hydrogel, possibly due to the increased intrinsic viscosity brought about by the formation of quinone in A35 MO10. This indicates that the significant enhancement of the adhesive strength of A35 MO is partly due to the adhesive effect between the dopa structure formed after tyrosinase modification and the interface.

[0206] To test the rheological properties using a rheometer, take 500 mL of each gel sample and spread it evenly on the surface of the cp25-2 rotor of the rheometer. Before testing, use a scraper to remove the excess material around the sample.

[0207] (1) Amplitude scan

[0208] Setting conditions: Strain (deformation) step change (CSD, logarithmic coordinates): strain amplitude γ = 0.01~100%, oscillation angular frequency ω = 10 rad / s.

[0209] like Figure 18 As shown, the linear viscoelastic region and viscoelasticity at an angular frequency of 10 rad / s of ASP and A35 MO were measured using the amplitude scanning mode of a rheometer. From the strain amplitude, the linear viscoelastic region endpoint of ASP is approximately 1%, while that of A35 MO exceeds 10%. Furthermore, it can be observed that the storage modulus of A35 MO within the linear viscoelastic region is an order of magnitude higher than that of ASP.

[0210] Therefore, A35 MO exhibits improved toughness and elasticity compared to ASP. It can be concluded that the tyrosinase-modified AS-Mefp3-P and AS-Mefp5-P mixed hydrogels, compared to ASP, not only undergo molecular-level changes, but also, due to the introduction of Mefp-3 and Mefp-5 and their interaction, the hydrogel as a whole is more tough and elastic. This may be because the cross-linking structure of A35 MO with the original ASP has significantly changed, becoming more compact, thus further improving the material properties. This conclusion is consistent with... Figure 17 The conclusions shown in the SEM experiments are consistent with those of the experiments.

[0211] (2) Frequency scanning

[0212] Setting conditions: frequency scan (CSD, logarithmic distribution), oscillation angular frequency ω = 100 ~ 0.01 rad / s, strain amplitude γ = 1% (strain within the LVE range must be used, which can be obtained from amplitude scan experiments).

[0213] The results are as follows Figure 19 As shown.

[0214] To further investigate the structural properties of A35 MO, samples were prepared by freezing the hydrogel sample in liquid nitrogen and then allowing it to stand in a lyophilizer. The internal structures of ASP and A35 MO samples were then observed using SEM. The main steps are as follows:

[0215] (1) After freezing the hydrogel sample with liquid nitrogen, place it in a freeze dryer for 48 hours.

[0216] (2) The freeze-dried sample was adhered to the sample detection stage with conductive adhesive to expose the cross-section, and then gold-plated for imaging characterization. The gold sputtering instrument was model SBC-12. The experiment was conducted using a SUPRA 55SAPPHIRE scanning electron microscope (Zeiss) with an operating voltage of 2kV.

[0217] like Figure 17 As shown, the pore size of the ASP sample after freeze-drying is approximately 5.29 μm, while that of A35 MO is approximately 1.86 μm. The pore size of the ASP sample is approximately three times that of A35 MO. This suggests that the degree of cross-linking formed by protein molecules in A35 MO is greater than that in the ASP sample. This cross-linking may be due to factors such as the dopa interaction in the tyrosinase-modified AS-Mefp3-P and AS-Mefp5-P proteins, leading to an increase in cross-linking density. This increased cross-linking density enhances the intrinsic viscosity of A35 MO, providing a second explanation for its improved adhesiveness.

[0218] The main steps of the bone adhesion test are as follows:

[0219] (1) Take 5 mg of 250 mg / mL hydrogel sample, apply it to the bottom of a petri dish with a coating area of ​​1 cm × 2 cm, and incubate it in a 37°C incubator for 15 min.

[0220] (2) Take 10 mg of the same type of hydrogel sample, spread it evenly on the bottom surface of the bovine bone, and incubate it in a 37°C incubator for 1 h.

[0221] (3) Pour 20 mL of deionized water into the culture dish.

[0222] (4) Lift one end of the cow bone so that the water-containing culture dish that is stuck to it is completely suspended in the air, and measure the time required for the culture dish and the cow bone to completely separate in this state.

[0223] like Figure 15 As shown, by evenly spreading the same mass of hydrogel between the petri dish and the bovine bone, and incubating at 37°C for 1 hour, 20g of deionized water was injected into the petri dish. One end of the bovine bone was then lifted, so that the water-containing petri dish, which was adhered to it, was completely suspended in mid-air (as shown). Figure 15 As shown in b), the time required for the culture dish and bovine bone to completely separate under these conditions was measured (this can be considered as the time required for the hydrogel to completely separate from the bovine bone). The experiment showed that the time required after applying the ASP hydrogel was 50 seconds, while that of A35 MO was 7 minutes. This further demonstrates that A35 MO exhibits stronger underwater adhesion capabilities.

[0224] To further explore the feasibility of underwater bonding with A35 MO, experiments were conducted to test its bonding effect on different material substrates (such as bone and glass slides). A35 MO was uniformly applied to the fractured bone and glass slides, and then immediately immersed in water. The results are as follows: Figure 25 As shown, this demonstrates that the A35 MO can maintain its underwater bond for at least 8 hours for both bone and glass bonding. This experiment illustrates the applicability of A35 MO to various materials.

[0225] The self-healing experiment mainly involves the following steps:

[0226] (1) Take a hydrogel sample onto a glass slide and cut it open from the middle with a sharp blade.

[0227] (2) Place the two cut hydrogels close together and record the time for complete self-healing.

[0228] like Figure 21 Images (a) to (e) illustrate the process of the hydrogel from intact to fragmented to fused together. Within approximately 2 minutes, A35 MO can recover from its damaged state to its intact state. Its self-healing properties likely stem from two aspects: firstly, the physical cross-linking within A35 MO; and secondly, the intermolecular hydrogen bonding between DOPA molecules in AS-Mefp3-P and AS-Mefp5-P. Studies have shown that this bonding can still occur even in water, as hydrogen bonding between DOPA molecules is more thermodynamically efficient than between DOPA and water.

[0229] Figure 22 As shown in Figure a, A35 MO was filled into a single piece of porous polyurethane material. This material was then cut in half. The two halves were brought close together, ensuring full contact between the A35 MO. After several minutes, due to the self-healing properties of A35 MO, the material returned to its original integrity, and it did not detach even when one end was picked up with tweezers. This experiment provides a new approach to the application of underwater adhesive materials, enabling fractured or damaged materials loaded with novel underwater adhesive materials to possess self-repairing capabilities.

[0230] The injectability of underwater adhesives is crucial for subsequent applications. To investigate whether A35 MO possesses injectability, 3D printing was used for verification. The main steps were as follows: a hydrogel sample was placed in a printing syringe, using a printing needle with an inner diameter of 21 mm and an outer diameter of 42 mm. The 3D printing instrument parameters were set to a system pressure of 700 kPa and a movement speed of 5 mm / s.

[0231] like Figure 23 Figure a shows the state of A35 MO when picked up with a spatula; it appears as a thin thread. Figure 23b shows the pattern formed by 3D printing A35 MO, where the side lengths of the hexagons are 3mm, 5mm, and 7mm from the inside out, and the diameters of the circles are 3mm, 5mm, and 7mm from the inside out. Experiments demonstrate that A35 MO was successfully printed, and the printed material is uniform, regular, and injectable. Based on the successful 3D printing demonstration in the experiment, this material has the potential to serve as an adhesive cell scaffold.

[0232] Figure 24 Examples of A35 MO's injectable properties include... Figure 24 Figure a shows an agarose gel, with the central portion missing. Figure 24 As shown in b, A35 MO was injected into the missing part using a 3D printer, and then three glass spheres were filled into the missing part. As shown in 24c, the material was inverted, and the spheres adhered tightly to the agarose gel through the A35 MO and did not fall off.

[0233] Example 8

[0234] An experiment investigating the fusion process of AS-Mefp3-P and AS-Mefp5-P:

[0235] The research objective of this paper is to improve the overall underwater adhesion of the material by simultaneously enhancing its intrinsic adhesion and interfacial adhesion. Based on the results of the above embodiments, the enhanced intrinsic adhesion of A35 MO is due to the self-assembly properties of the leucine zipper on the one hand, and may also originate from the interaction between AS-Mefp3-P and AS-Mefp5-P on the other hand.

[0236] To further verify whether there is an interaction between AS-Mefp3-P and AS-Mefp5-P modified with tyrosinase, the following experiments were conducted in this invention:

[0237] (1) The constructed AS-Spytag-Mefp3-P and AS-Snooptag-Mefp5-P were subjected to protein expression, separation and purification experiments in the manner described above. The obtained AS-Spytag-Mefp3-P was mixed with an appropriate amount of McCherry-Spycatcher solution (the sequence of McCherry-Spycatcher is that the C-terminus of McCherry is directly linked to the N-terminus of Spycatcher, the amino acid sequence of Spycatcher is shown in SEQ ID No. 16, and the amino acid sequence of McCherry is shown in SEQ ID No. 18). AS-Snooptag-Mefp5-P was mixed with an appropriate amount of GFP-Snoopcatcher (the sequence of GFP-Snoopcatcher is that the C-terminus of GFP is directly linked to the N-terminus of Snoopcatcher, the amino acid sequence of Snoopcatcher is shown in SEQ ID No. 17, and the amino acid sequence of GFP is shown in SEQ ID No. 19), and gelled in the same manner described above.

[0238] (2) The AS-Spytag-Mefp3-P hydrogel (hereinafter referred to as ASpy) containing Cherry-Spycatcher was cut in half from the middle, and the same operation was performed on the AS-Snooptag-Mefp5- hydrogel (hereinafter referred to as ASnoop) containing GFP-Snoopcatcher. Then, the half of the ASpy hydrogel and the half of the Asnoop hydrogel were brought close together, and the phenomenon of their contact interface was observed under a fluorescence microscope.

[0239] The results are as follows Figure 20 As shown in Figures 20d to e, some changes occurred at the interface where Aspy and Asnoop were close together. Within the ellipse without arrows, it is evident that Aspy and Asnoop fused, as reflected in the deepening yellow hue resulting from the fusion of their green and red fluorescence. Within the ellipse with arrows, it is visible that the distance between the fixed points of Aspy and Asnoop decreased. This dynamic fusion process was observed within just 10 minutes. Based on these experiments, it can be inferred that there may be an interaction between AS-Mefp3-P and AS-Mefp5-P. Further experiments at longer time scales can be conducted to verify this.

[0240] In summary, this invention effectively overcomes the various shortcomings of the prior art and has high industrial application value.

[0241] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention. sequence list <110> ShanghaiTech University <120> An injectable self-healing underwater protein and its uses <160> 34 <170> SIPOSequenceListing 1.0 <210> 1 <211> 42 <212> PRT <213> Artificial Sequence A <400> 1 Ser Gly Asp Leu Glu Asn Glu Val Ala Gln Leu Glu Arg Glu Val Arg 1 5 10 15 Ser Leu Glu Asp Glu Ala Ala Glu Leu Glu Gln Lys Val Ser Arg Leu 20 25 30 Lys Asn Glu Ile Glu Asp Leu Lys Ala Glu 35 40 <210> 2 <211> 90 <212> PRT <213> Artificial Sequence S Fragment <400> 2 Ala Gly Ala Gly Ala Gly Pro Glu Gly Ala Gly Ala Gly Ala Gly Pro 1 5 10 15 Glu Gly Ala Gly Ala Gly Ala Gly Pro Glu Gly Ala Gly Ala Gly Ala 20 25 30 Gly Pro Glu Gly Ala Gly Ala Gly Ala Gly Pro Glu Gly Ala Gly Ala 35 40 45 Gly Ala Gly Pro Glu Gly Ala Gly Ala Gly Ala Gly Pro Glu Gly Ala 50 55 60 Gly Ala Gly Ala Gly Pro Glu Gly Ala Gly Ala Gly Ala Gly Pro Glu 65 70 75 80 Gly Ala Gly Ala Gly Ala Gly Pro Glu Gly 85 90 <210> 3 <211> 42 <212> PRT <213> Artificial Sequence P fragment <400> 3 Ala Pro Gln Met Leu Arg Glu Leu Gln Glu Thr Asn Ala Ala Leu Gln 1 5 10 15 Asp Val Arg Glu Leu Leu Arg Gln Gln Val Lys Glu Ile Thr Phe Leu 20 25 30 Lys Asn Thr Val Met Glu Ser Asp Ala Ser 35 40 <210> 4 <211> 48 <212> PRT <213> Artificial Sequence Mefp-3 <400> 4 Ala Asp Tyr Tyr Gly Pro Asn Tyr Gly Pro Pro Arg Arg Tyr Gly Gly 1 5 10 15 Gly Asn Tyr Asn Arg Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr 20 25 30 Lys Gly Trp Asn Asn Gly Trp Asn Arg Gly Arg Arg Gly Lys Tyr Trp 35 40 45 <210> 5 <211> 90 <212> PRT <213> Artificial Sequence Mefp-5 <400> 5 Ala Gly Ala Gly Ala Gly Pro Glu Gly Ala Gly Ala Gly Ala Gly Pro 1 5 10 15 Glu Gly Ala Gly Ala Gly Ala Gly Pro Glu Gly Ala Gly Ala Gly Ala 20 25 30 Gly Pro Glu Gly Ala Gly Ala Gly Ala Gly Pro Glu Gly Ala Gly Ala 35 40 45 Gly Ala Gly Pro Glu Gly Ala Gly Ala Gly Ala Gly Pro Glu Gly Ala 50 55 60 Gly Ala Gly Ala Gly Pro Glu Gly Ala Gly Ala Gly Ala Gly Pro Glu 65 70 75 80 Gly Ala Gly Ala Gly Ala Gly Pro Glu Gly 85 90 <210> 6 <211> 46 <212> PRT <213> Artificial Sequence Mgfp-3 <400> 6 Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly 1 5 10 15 Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp 20 25 30 Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 <210> 7 <211> 76 <212> PRT <213> Artificial Sequence Mgfp-5 <400> 7 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His 1 5 10 15 Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr 20 25 30 Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys 35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg 50 55 60 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser 65 70 75 <210> 8 <211> 45 <212> PRT <213> Artificial Sequence mfp‑3s <400> 8 Gly Tyr Gly Tyr Asp Leu Gly Tyr Asn Ala Pro Trp Pro Tyr Asn Asn 1 5 10 15 Gly Tyr Tyr Gly Tyr Asn Gly Tyr Asn Gly Tyr His Gly Arg Tyr Gly 20 25 30 Trp Asn Lys Gly Trp Asn Asn Gly Pro Trp Gly Gly Tyr 35 40 45 <210> 9 <211> 25 <212> PRT <213> Artificial Sequence mfp3s‑pep <400> 9 Gly Tyr Asp Gly Tyr Asn Trp Pro Tyr Gly Tyr Asn Gly Tyr Arg Tyr 1 5 10 15 Gly Trp Asn Lys Gly Trp Asn Gly Tyr 20 25 <210> 10 <211> 14 <212> PRT <213> Artificial Sequence A1 Fragment <220> <221> VARIANT <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Glu, or Lys. <220> <221> VARIANT <222> (7)..(7) <223> The 'Xaa' at location 7 stands for Glu, or Lys. <220> <221> VARIANT <222> (12)..(12) <223> The 'Xaa' at location 12 stands for Glu, or Lys. <220> <221> VARIANT <222> (14)..(14) <223> The 'Xaa' at location 14 stands for Glu, or Lys. <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (7)..(7) <223> The 'Xaa' at location 7 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (12)..(12) <223> The 'Xaa' at location 12 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (14)..(14) <223> The 'Xaa' at location 14 stands for Gln, Arg, Pro, or Leu. <400> 10 Ser Gly Asp Leu Xaa Asn Xaa Val Ala Gln Leu Xaa Arg Xaa 1 5 10 <210> 11 <211> 14 <212> PRT <213> Artificial Sequence A1 Fragment <220> <221> VARIANT <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Glu, or Lys. <220> <221> VARIANT <222> (7)..(7) <223> The 'Xaa' at location 7 stands for Glu, or Lys. <220> <221> VARIANT <222> (12)..(12) <223> The 'Xaa' at location 12 stands for Glu, or Lys. <220> <221> VARIANT <222> (14)..(14) <223> The 'Xaa' at location 14 stands for Glu, or Lys. <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (7)..(7) <223> The 'Xaa' at location 7 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (12)..(12) <223> The 'Xaa' at location 12 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (14)..(14) <223> The 'Xaa' at location 14 stands for Gln, Arg, Pro, or Leu. <400> 11 Val Arg Ser Leu Xaa Asp Xaa Ala Ala Glu Leu Xaa Gln Xaa 1 5 10 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence A1 Fragment <220> <221> VARIANT <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Glu, or Lys. <220> <221> VARIANT <222> (7)..(7) <223> The 'Xaa' at location 7 stands for Glu, or Lys. <220> <221> VARIANT <222> (12)..(12) <223> The 'Xaa' at location 12 stands for Glu, or Lys. <220> <221> VARIANT <222> (14)..(14) <223> The 'Xaa' at location 14 stands for Glu, or Lys. <220> <221> UNSURE <222> (5)..(5) <223> The 'Xaa' at location 5 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (7)..(7) <223> The 'Xaa' at location 7 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (12)..(12) <223> The 'Xaa' at location 12 stands for Gln, Arg, Pro, or Leu. <220> <221> UNSURE <222> (14)..(14) <223> The 'Xaa' at location 14 stands for Gln, Arg, Pro, or Leu. <400> 12 Val Ser Arg Leu Xaa Asn Xaa Ile Glu Asp Leu Xaa Ala Xaa Ile 1 5 10 15 <210> 13 <211> 37 <212> PRT <213> γ52−88KI fragment (Artificial Sequence) <400> 13 Ile Asp Phe Ile Ser Thr Tyr Ile Thr Lys Ile Asp Lys Lys Ile Gln 1 5 10 15 Ser Ile Glu Asp Ile Ile His Gln Ile Glu Asn Lys Ile Ser Glu Ile 20 25 30 Lys Gln Leu Ile Lys 35 <210> 14 <211> 13 <212> PRT <213> Artificial Sequence Spytag <400> 14 Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys 1 5 10 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence Snooptag <400> 15 Gly Lys Leu Gly Asp Ile Glu Phe Ile Lys Val Asn Lys Gly Tyr 1 5 10 15 <210> 16 <211> 113 <212> PRT <213> Artificial Sequence spycatcher <400> 16 Val Asp Thr Leu Ser Gly Leu Ser Ser Glu Gln Gly Gln Ser Gly Asp 1 5 10 15 Met Thr Ile Glu Glu Asp Ser Ala Thr His Ile Lys Phe Ser Lys Arg 20 25 30 Asp Glu Asp Gly Lys Glu Leu Ala Gly Ala Thr Met Glu Leu Arg Asp 35 40 45 Ser Ser Gly Lys Thr Ile Ser Thr Trp Ile Ser Asp Gly Gln Val Lys 50 55 60 Asp Phe Tyr Leu Tyr Pro Gly Lys Tyr Thr Phe Val Glu Thr Ala Ala 65 70 75 80 Pro Asp Gly Tyr Glu Val Ala Thr Ala Ile Thr Phe Thr Val Asn Glu 85 90 95 Gln Gly Gln Val Thr Val Asn Gly Lys Ala Thr Lys Gly Asp Ala His 100 105 110 Ile <210> 17 <211> 112 <212> PRT <213> Artificial Sequence Snoopcatcher <400> 17 Lys Pro Leu Arg Gly Ala Val Phe Ser Leu Gln Lys Gln His Pro Asp 1 5 10 15 Tyr Pro Asp Ile Tyr Gly Ala Ile Asp Gln Asn Gly Thr Tyr Gln Asn 20 25 30 Val Arg Thr Gly Glu Asp Gly Lys Leu Thr Phe Lys Asn Leu Ser Asp 35 40 45 Gly Lys Tyr Arg Leu Phe Glu Asn Ser Glu Pro Ala Gly Tyr Lys Pro 50 55 60 Val Gln Asn Lys Pro Ile Val Ala Phe Gln Ile Val Asn Gly Glu Val 65 70 75 80 Arg Asp Val Thr Ser Ile Val Pro Gln Asp Ile Pro Ala Thr Tyr Glu 85 90 95 Phe Thr Asn Gly Lys His Tyr Ile Thr Asn Glu Pro Ile Pro Pro Lys 100 105 110 <210> 18 <211> 237 <212> PRT <213> Artificial Sequence Mcherry <400> 18 Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met 1 5 10 15 Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu 20 25 30 Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala 35 40 45 Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile 50 55 60 Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His Pro 65 70 75 80 Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe Lys 85 90 95 Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr 100 105 110 Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys Leu 115 120 125 Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr 130 135 140 Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala 145 150 155 160 Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly His 165 170 175 Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln 180 185 190 Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser His 195 200 205 Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg 210 215 220 His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Ala Gly 225 230 235 <210> 19 <211> 240 <212> PRT <213> Artificial Sequence Gfp <400> 19 Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ala Gly 225 230 235 240 <210> 20 <211> 585 <212> DNA <213> Artificial Sequence A-S-P plasmid <400> 20 atgtctggtg acctggaaaa cgaagttgcg cagctggaac gtgaagttcg ttctctggaa 60 gacgaagcgg cggaactgga acagaaagtt tctcgtctga aaaacgaaat cgaagacctg 120 aaagcggaac catgggcagg tgccggtgca ggtccggag gtgcaggtgc aggtgcaggt 180 ccggaaggtg caggtgcagg tgcaggtccg gagggtgccg gtgcaggtgc aggtcctgaa ggtgccggtg caggtgcagg tccggaaggt gcaggtgcag gtgccggtcc tgaaggtgcc 300 ggtgcaggtg caggtcctga aggtgccggt gcaggtgccg gtcctgaggg tgcaggtgca 360 ggtgccggcc ctgaaggtgc aggtgcaggt gcaggtccgg aaggtggatc caagcttggc 420 ggtggcggta gcgctccgca gatgctgcgt gaactgcagg aaaccaacgc tgctctgcag 480 gacgttcgtg aactgctgcg tcagcaggtt aaagaaatca ccttcctgaa aaacaccgtt 540 atggaatctg acgcttctct cgagcaccac caccaccacc actga <210> 21 <211> 79 <212> DNA <213> Material A‐S‐P‐F Conjugation (Artificial Sequence) <400> 21 60. tacatatgtc tggtgacctg gaaacgaag ttgcgcagct ggaacgtgaa gttcgttctc tggaagacga agcggcgga <210> 22 <211> 84 <212> DNA <213> Material A‐S‐P‐R Conjugation (Artificial Sequence) <400> 22 gcccatggtt ccgctttcag gtcttcgatt tcgtttttca gacgagaaac tttctgttcc agttccgccg cttcgtcttc caga <210> 23 <211> 729 <212> DNA <213> Enzyme A‐S‐Mefp3‐P (Artificial Sequence) <400> 23 atgtctggtg acctggaaaa cgaagttgcg cagctggac gtgaagttcg ttctctggaa 120. gacgaagcgg cggaactgga acagaaagtt tctcgtctga aaaacgaaat cgaagacctg aaagcggaac catgggcagg tgccggtgca ggtccggag gtgcaggtgc aggtgcaggt 180 ccggaaggtg caggtgcagg tgcaggtccg gagggtgccg gtgcaggtgc aggtcctgaa ggtgccggtg caggtgcagg tccggaaggt gcaggtgcag gtgccggtcc tgaaggtgcc 300 ggtgcaggtg caggtcctga aggtgccggt gcaggtgccg gtcctgaggg tgcaggtgca 360 ggtgccggcc ctgaaggtgc aggtgcaggt gcaggtccgg aaggtggatc cgccgattat 420 tatggcccga attatggtcc gccgcgtcgc tacggtggcg gcaactataa ccgctacaat cgctatggcc gccgctatgg tggctaaa ggctggaaca acggctgga tcgcggtcgc 540 cgcggtaaat attggaagct tggcggtggc ggtagcgctc cgcagatgct gcgtgaactg caggaacca acgctgctct gcaggacgtt cgtgaactgc tgcgtcagca ggttaaaga atcaccttcc tgaaaaacac cgttatggaa tctgacgctt ctctcgagca ccaccaccac caccactga 729 <210> 24 <211> 26 <212> DNA <213> A‐S‐Mefp3‐P‐F Protozoan (Artificial Sequence) <400> 24 ggatccgccg attack cccgaa <210> 25 <211> 25 <212> DNA <213> A‐S‐Mefp3‐P‐F Protozoan (Artificial Sequence) <400> 25 aagcttccaa tatttaccgc ggcga <210> 26 <211> 807 <212> DNA <213> Protein A‐S‐Mefp5‐P (Artificial Sequence) <400> 26 atgtctggtg acctggaaaa cgaagttgcg cagctggac gtgaagttcg ttctctggaa 120. gacgaagcgg cggaactgga acagaaagtt tctcgtctga aaaacgaaat cgaagacctg aaagcggaac catgggcagg tgccggtgca ggtccggag gtgcaggtgc aggtgcaggt 180 ccggaaggtg caggtgcagg tgcaggtccg gagggtgccg gtgcaggtgc aggtcctgaa ggtgccggtg caggtgcagg tccggaaggt gcaggtgcag gtgccggtcc tgaaggtgcc 300 ggtgcaggtg caggtcctga aggtgccggt gcaggtgccg gtcctgaggg tgcaggtgca 360 ggtgccggcc ctgaaggtgc aggtgcaggt gcaggtccgg aaggtggatc cagcagtgaa 420 480. gcggctta cccgggtac gcgtaccatt accacagcgg tggtagttat 540. catggaagcg gctaccacgg tggctacaaa ggcaagtact acggtaaggc caagaatac 600. 600. 600. 600. 600. 600. 600. 600. 600. 600 cgtaaagggt our father tggtggctct tcaaagcttg gcggtggcgg tagcgctccg 660 cagatgctgc gtgaactgca ggaaaccaac gctgctctgc aggacgttcg tgaactgctg cgtcagcagg ttaagaat caccttcctg aaaaacaccg ttatggaatc tgacgcttct ctcgagcacc 807. ctcgagcacc <210> 27 <211> 26 <212> DNA <213> A‐S‐Mefp5‐P‐F Protozoan (Artificial Sequence) <400> 27 ggatccagca gtgaagaata taaagg <210> 28 <211> 28 <212> DNA <213> A‐S‐Mefp5‐P‐R group (Artificial Sequence) <400> 28 aagctttgaa gagccaccat today <210> 29 <211> 768 <212> DNA <213> Encoding A‐S‐Spytag‐Mefp3‐P (Artificial Sequence) <400> 29 atgtctggtg acctggaaaa cgaagttgcg cagctggac gtgaagttcg ttctctggaa 120. gacgaagcgg cggaactgga acagaaagtt tctcgtctga aaaacgaaat cgaagacctg aaagcggaac catgggcagg tgccggtgca ggtccggag gtgcaggtgc aggtgcaggt 180 ccggaaggtg caggtgcagg tgcaggtccg gagggtgccg gtgcaggtgc aggtcctgaa ggtgccggtg caggtgcagg tccggaaggt gcaggtgcag gtgccggtcc tgaaggtgcc 300 ggtgcaggtg caggtcctga aggtgccggt gcaggtgccg gtcctgaggg tgcaggtgca 360 ggtgccggcc ctgaaggtgc aggtgcaggt gcaggtccgg aaggtggatc cgcgcacatc 420 gttatggtcg atgcatata acccaccaaa gccgattatt atggcccgaa ttatggtccg ccgcgtcgct acggtggcgg caactatac cgctacaatc gctatggccg ccgctatggt ggctaaag gctggaacaa cggctggaat cgcggtcgcc gcggtaaata ttggaagctt ggcggtggcg gtagcgctcc gcagatgctg cgtgaactgc attack cgctgctctg caggacgttc gtgaactgct gcgtcagcag gttaaagaaa tcaccttcct gaaaaacacc gttatggaat ctgacgcttc tctcgagcac caccaccacc accactga <210> 30 <211> 65 <212> DNA <213> Material A‐S‐Spytag‐Mefp3‐P‐F Protozoan (Artificial Sequence) <400> 30 ggatccgcgc acatcgttat ggtcgatgca tataaccca ccaaagccga ttattatggc ccgaa 65 <210> 31 <211> 28 <212> DNA <213> Material A‐S‐Spytag‐Mefp3‐P‐R Enzyme (Artificial Sequence) <400> 31 aagcttccaa tatttaccgc ggcgaccg <210> 32 <211> 852 <212> DNA <213> Encoding A‐S‐Snooptag‐Mefp5‐P (Artificial Sequence) <400> 32 atgtctggtg acctggaaaa cgaagttgcg cagctggac gtgaagttcg ttctctggaa 120. gacgaagcgg cggaactgga acagaaagtt tctcgtctga aaaacgaaat cgaagacctg aaagcggaac catgggcagg tgccggtgca ggtccggag gtgcaggtgc aggtgcaggt 180 ccggaaggtg caggtgcagg tgcaggtccg gagggtgccg gtgcaggtgc aggtcctgaa ggtgccggtg caggtgcagg tccggaaggt gcaggtgcag gtgccggtcc tgaaggtgcc 300 ggtgcaggtg caggtcctga aggtgccggt gcaggtgccg gtcctgaggg tgcaggtgca 360 ggtgccggcc ctgaaggtgc aggtgcaggt gcaggtccgg aaggtggatc cggaaaactg 420 ggggacatcg aattcatcaa agtaaacaaa ggttacagca gtgaagaata taaaggcggc tattacccgg gtaacgcgta ccattaccac agcggtggta gttatcatgg aagcggctac cacggtggct acaaaggcaa gtactacggt aaggccaaga aatactacta caatacaaa aattcaggca agtacaata tctgaagaaa gcgcgcaaat accaccgtaa agggtataaa tactatggtg gctcttcaaa gcttggcggt ggcggtagcg ctccgcagat gctgcgtgaa 720 ctgcaggaa ccaacgctgc tctgcaggac gttcgtgac tgctgcgtca gcaggttaaa gaatcacct tcctgaaaaa caccgttatg gaatctgacg cttctctcga gcaccaccac 852 caccaccact <210> 33 <211> 71 <212> DNA <213> Citation A‐S‐Snooptag‐Mefp5‐P‐F Protozoan (Artificial Sequence) <400> 33 ggatccgga aactggggga catcgaattc atcaaagtaa acaaaggtta cagcagtgaa gathering g <210> 34 <211> 29 <212> DNA <213> Encoding A‐S‐Snooptag‐Mefp5‐P‐R Protozoan (Artificial Sequence) <400> 34 aagctttgaa gagccaccat agtatttat

Claims

1. A fusion protein comprising a mussel byssal protein fragment and a coiled-coil structure fragment, wherein the coiled-coil structure fragment is a leucine zipper fragment; the leucine zipper fragment comprises, from the N-terminus to the C-terminus, a fragment A, a fragment S, and a fragment P; the mussel byssal protein fragment is inserted into the leucine zipper fragment; the mussel byssal protein is selected from mefp-3 or mefp-5, the amino acid sequence of mefp-3 is shown in SEQ ID No. 4, and the amino acid sequence of mefp-5 is shown in SEQ ID No. 5; the fusion protein is modified with tyrosinase; the fusion protein is composed of, from the N-terminus to the C-terminus, a fragment A, a fragment S, a mussel byssal protein fragment, and a fragment P, the amino acid sequence of the fragment A is shown in SEQ ID No. 1, the amino acid sequence of the fragment S is shown in SEQ ID No. 2, and the amino acid sequence of the fragment P is shown in SEQ ID No.

3.

2. An isolated polynucleotide encoding the fusion protein as described in claim 1.

3. A construct comprising the isolated polynucleotide of claim 2.

4. An expression system comprising an exogenous polynucleotide as described in claim 2 integrated into a construct or genome as described in claim 3.

5. A method for preparing the fusion protein according to any one of claims 1-2, comprising: The expression system as described in claim 4 is cultured under conditions suitable for expressing the fusion protein.

6. The preparation method according to claim 5, characterized in that, The resulting fusion protein was modified with tyrosinase.

7. A self-assembling colloidal solution comprising the fusion protein as described in claim 1.

8. A colloid comprising the fusion protein as described in claim 1.

9. The colloid as described in claim 8, characterized in that, It also includes one or more of the following technical features: C1) The colloid is a hydrogel; C2) The content of fusion protein in the colloid is 70 mg / mL to 250 mg / mL; C3) The colloid is obtained by the self-assembly of the fusion protein; C4) The colloid is prepared from the self-assembled colloidal solution of claim 8; In the colloid described in C5), the mussel foot filament protein in the fusion protein is selected from a combination of mefp-3 and mefp-5.

10. Use of the fusion protein of claim 1, the self-assembling colloidal solution of claim 7, and the colloid of any one of claims 8-9 in the field of underwater adhesive preparation.

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