A method for self-assembling any recombinant protein into supramolecular colloids

Abstract

The present invention discloses a method for making any recombinant protein self-assemble into supramolecular colloid. Existing polypeptide hydrogels mainly rely on chemical synthesis of target polypeptides to form injectable hydrogels. However, chemical synthesis is expensive and difficult to apply on a large scale, and chemical synthesis can only be used to synthesize active short peptides with fewer sequences, and cannot synthesize macromolecular proteins with longer sequences. The present invention overcomes the above difficulties and can make any recombinant protein self-assemble into supramolecular hydrogel without relying on chemical synthesis. Specific method: The amyloid self-assembling short peptide Ure2 (1-80) is fused to any recombinant protein, and the obtained fusion protein will form nanofiber-like self-assembly aggregation at a concentration lower than 32 mg / mL, and will form supramolecular hydrogels with characteristics such as shear thinning effect and injectability when it is greater than or equal to 32 mg / mL. The formation of this hydrogel does not require any additional artificial interference, and only needs to be left to stand for a period of time to complete the gelation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for enabling any recombinant protein to self-assemble into supramolecular colloid. Background Art

[0002] Driving individual peptides into more ordered supramolecular structures, often called self-assembly, is a hallmark of natural biological systems. The self-assembly process is controlled by non-covalent physical interactions, such as hydrogen bonds, van der Waals forces, ionic interactions, hydrophobic interactions, etc. The accumulation of these physical interactions ultimately generates stable, ordered supramolecular structures, resulting in complex and unique material functions. Therefore, nanomaterials based on self-assembling short peptides are very promising materials in many biomedical and biotechnology fields, including tissue engineering, regenerative medicine, enzyme catalysis, biosensors and drug delivery.

[0003] Among these nanomaterials, self-assembled materials based on recombinant proteins have many advantages over traditional synthetic polymers or natural materials. For example, chemically synthesized polymers are limited in application by their inherent bioincompatibility, biodegradability, and the production of toxic byproducts. Although naturally derived protein materials such as collagen, laminin, and Matrigel have become the main components of 3D cell culture and tissue engineering scaffolds, these materials are not suitable for medical applications. First, the above materials are separated from living animal tissues, which may have potential adverse immune reactions and high infection risks. Matrigel is derived from mouse EHS sarcoma. At the same time, the quality of animal-derived materials usually varies from batch to batch, and may have adverse effects due to unknown cell signaling factors present in the materials. In contrast, self-assembled recombinant protein materials synthesized by microorganisms are particularly attractive, not only because they have excellent biodegradability, biocompatibility, and similarity to the extracellular matrix, but also because they have batch-stable homogeneity, sequence and functional programmability, low cost, and easy large-scale production.

[0004] So far, self-assembling recombinant protein materials mainly rely on chemical synthesis of short protein peptides with self-assembly functions, such as amphiphilic peptides, which are composed of alternating hydrophilic and hydrophobic amino acids and self-assemble into β-folded nanostructured hydrogels in solution. However, chemical synthesis is expensive and difficult to apply on a large scale; chemical synthesis is difficult to synthesize large molecular proteins, resulting in the current self-assembling recombinant protein gel materials mainly focusing on the self-assembly of small molecular active peptides into colloids, and no reports have been reported on the self-assembly of large molecular functional proteins into colloids. Therefore, how to reduce the production cost of self-assembling proteins and enable large molecular functional proteins to also achieve self-assembly into hydrogels is an urgent problem to be solved. Summary of the invention

[0005] To solve the above problems, including: 1) Currently, self-assembling recombinant protein materials mainly rely on chemical synthesis of short protein peptides with self-assembly function, which is expensive and difficult to apply on a large scale; 2) It is difficult to synthesize large molecular proteins by chemical synthesis, resulting in only small molecular active peptides self-assembling into hydrogels, while it is difficult to achieve self-assembly of large molecular functional proteins into hydrogels; this project provides a self-assembly gelation mechanism based on the amyloid self-assembling short peptide Ure2 (1-80), which can enable any large molecular protein to self-assemble into supramolecular nanomaterials such as nanofibers and hydrogels, and no additional artificial chemical synthesis of self-assembling short peptides is required.

[0006] A self-assembly gelation mechanism based on the amyloid self-assembly short peptide Ure2 (1-80). The self-assembly short peptide can enable any recombinant protein expressed by engineering bacteria to self-assemble into supramolecular nanomaterials such as nanofibers and hydrogels.

[0007] Furthermore, the self-assembling short peptide is the amyloid fiber Ure2 (1-80) in Saccharomyces cerevisiae, and its amino acid sequence is: MMNNNGNQVSNLSNALRQVNIGNRNSNTTTDQSNINFEFSTGVNNNNNNNSSSNNNNVQNNNSGRNGSQNNDNENNIKNT.

[0008] Furthermore, for the above amino acid sequence truncated from the C-terminus to the 16th amino acid, each short peptide mutant generated also has the property of self-assembling any recombinant protein into supramolecular nanomaterials such as nanofibers and hydrogels.

[0009] Furthermore, any amino acid substitution of no more than 10% in the above amino acid sequence will still not change the characteristics of the above short peptide self-assembly recombinant protein.

[0010] Furthermore, by fusing the self-assembling short peptide to the N-terminus or C-terminus of any recombinant protein through genetic engineering, the recombinant protein can be self-assembled into supramolecular materials such as nanofibers and hydrogels.

[0011] Furthermore, the recombinant protein fused with the self-assembling short peptide will form nanofiber self-assembling aggregates when the working concentration is lower than 32 mg / ml (such as 2-16 mg / ml). The working environment can be any aqueous solution, including pure water and various buffer solutions. The ambient temperature is not limited and can be carried out at 0°C-room temperature. The time for self-assembly to complete is shortened as the concentration of the fusion protein increases. At low concentrations (2 mg / ml), it takes about 2 days to complete the entire self-assembly. At higher concentrations (32 mg / ml), it takes about 5 hours to complete the entire self-assembly, at which time the supramolecular hydrogel has been formed.

[0012] Furthermore, the recombinant protein fused with the self-assembling short peptide will form a supramolecular hydrogel with the characteristics of shear thinning effect and injectability when the working concentration is greater than or equal to 32 mg / mL. The formation of this hydrogel does not require any additional artificial interference, such as adjusting the pH, adding chemical cross-linking agents, etc.; the working environment can be any aqueous solution, including pure water and various buffer solutions, and the ambient temperature is not limited, and can be carried out at 0°C-room temperature; at a lower concentration (32 mg / ml), it takes about 5 hours to complete the formation of the hydrogel, and at a high concentration (75 mg / ml), it only takes 30 minutes to complete the gelation.

[0013] Furthermore, different recombinant proteins fused with self-assembling short peptides can be mixed in any proportion when the working concentration is greater than or equal to 32 mg / mL, and form a supramolecular composite hydrogel with characteristics such as shear thinning effect and injectability. The formation of this composite hydrogel does not require any additional artificial interference, such as adjusting pH, adding chemical cross-linking agents, etc.; the working environment can be any aqueous solution, including pure water and various buffer solutions, and the ambient temperature is not limited, and can be carried out at 0°C-room temperature; at a lower concentration (32 mg / ml), it takes about 5 hours to complete the formation of the hydrogel, and at a high concentration (75 mg / ml), it only takes 30 minutes to complete the gelation.

[0014] Beneficial effects of the present invention:

[0015] The present invention obtains an amyloid self-assembling short peptide Ure2 (1-80) by screening from saccharomyces cerevisiae, connects the short peptide sequence or its mutant to any recombinant protein by fusion method, and the obtained fusion protein forms supramolecular hydrogel or supramolecular nanomaterials such as nanofibers with characteristics such as shear thinning effect and injectability. The formation of the hydrogel does not require additional artificial chemical synthesis of the self-assembling short peptide, which reduces the production cost of supramolecular nanomaterials, and does not require any additional artificial interference, such as adjusting pH, adding chemical cross-linking agents, etc., and only needs to stand to complete the gelation. The recombinant protein self-assembly mechanism developed by the present invention will have broad applications in the fields of injectable gel, tissue engineering, drug delivery, enzyme immobilization, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the aggregate formed by the green fluorescent protein (Ure2(1-80)-EGFP) fused with the self-assembling short peptide Ure2(1-80) at low concentration (2 mg / ml);

[0017] Figure 2It is the aggregate formed by the green fluorescent protein (Ure2(jieduan8)-EGFP) fused with the short peptide mutant Ure2(jieduan8) with 8 amino acids truncated in the C segment at low concentration (2mg / ml);

[0018] Figure 3 It is the aggregate formed by the green fluorescent protein (Ure2(jieduan)-EGFP) fused with the short peptide mutant Ure2(jieduan) with 16 amino acids truncated in the C segment at low concentration (2mg / ml);

[0019] Figure 4 It is the aggregate formed by the green fluorescent protein (Ure2(tubian)-EGFP) fused with the short peptide mutant Ure2(tubian) with 4 random amino acids mutated at low concentration (2mg / ml);

[0020] Figure 5 It is the aggregate formed by the green fluorescent protein (Ure2(tubian)-EGFP) fused with the short peptide mutant Ure2(tubian) with random mutation of 8 amino acids at low concentration (2mg / ml);

[0021] Figure 6 The formation efficiency of nanofibers fused with fluorescent proteins and different self-assembling short peptides;

[0022] Figure 7 The nanofiber structure formed by Ure2(1-80)-EGFP fusion protein under transmission electron microscopy;

[0023] Figure 8 The nanofiber structure formed by Ure2(jieduan)-EGFP fusion protein under transmission electron microscopy;

[0024] Fig. 9 The nanofiber structure formed by Ure2 (tubian)-EGFP fusion protein under transmission electron microscopy;

[0025] Fig.10 The active self-assembled aggregates formed by Ure2(1-80)-EGFP at different concentrations;

[0026] Fig.11 Thioflavin T (ThT) was used to detect the formation of nanofibers of Ure2(1-80)-EGFP at different concentrations;

[0027] Fig.12 The nanohydrogels formed by Ure2(1-80)-EGFP and Ure2(1-80)-mCherry at low concentration (32 mg / ml);

[0028] Fig.13 It is a supramolecular composite hydrogel formed by equal ratios of Ure2(1-80)-EGFP and Ure2(1-80)-mCherry at high concentration (75 mg / ml);

[0029] Fig.14 It is a supramolecular composite hydrogel formed by Ure2(1-80)-EGFP and Ure2(1-80)-mCherry at different ratios at low concentration (32 mg / ml);

[0030] Fig.15 The rheological properties of supramolecular hydrogels formed by different concentrations of Ure2(1-80)-EGFP;

[0031] Fig.16 It is a supramolecular hydrogel formed by the green fluorescent protein (Ure2(jieduan8)-EGFP) fused with a short peptide mutant Ure2(jieduan8) with 8 amino acids truncated in the C segment at a low concentration (32mg / ml);

[0032] Fig.17 It is a supramolecular hydrogel formed by the green fluorescent protein (Ure2(jieduan)-EGFP) fused with a short peptide mutant Ure2(jieduan) with 16 amino acids truncated in the C segment at a high concentration (75mg / ml);

[0033] Fig.18 It is a supramolecular hydrogel formed by the green fluorescent protein (Ure2(tubian)-EGFP) fused with a short peptide mutant Ure2(tubian) with four amino acids randomly mutated at a low concentration (32 mg / ml);

[0034] Fig.19 The present invention is a supramolecular hydrogel formed by the green fluorescent protein (Ure2(tubian)-EGFP) fused with a short peptide mutant Ure2(tubian) in which 8 amino acids are randomly mutated at a high concentration (75 mg / ml). DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0036] The scheme involved in the present invention is as follows:

[0037] The present invention provides a method for self-assembling any recombinant protein into a supramolecular colloid, comprising the following steps:

[0038] Fusing any protein with an amyloid short peptide or its mutant, and then placing one or more recombinant proteins in an aqueous solution for self-assembly;

[0039] Among them, the amino acid sequence of the amyloid short peptide is:

[0040] MMNNNGNQVSNLSNALRQVNIGNRNSNTTTDQSNINFEFSTGVNNN NNNNSSSNNNNVQNNNSGRNGSQNNDNENNIKNT.

[0041] Furthermore, the amyloid peptide mutant is any one of the following:

[0042] (a) The amyloid peptide is truncated from the C-terminus to any of the 1st to 16th amino acids;

[0043] (b) The amyloid peptide undergoes no more than 10% amino acid substitution.

[0044] Among the above mutants, no matter whether they are obtained by truncation or amino acid substitution, the properties of the above recombinant protein will not be changed. Each short peptide mutant generated also has the property of self-assembling any recombinant protein into a hydrogel.

[0045] Furthermore, the amyloid peptide or its mutant is fused to the N-terminus or C-terminus of any protein.

[0046] Furthermore, one or more recombinant proteins are placed in an aqueous solution and allowed to stand to obtain a hydrogel.

[0047] Further, let it stand for 30 minutes to 5 hours.

[0048] Furthermore, the concentration of the recombinant protein in the aqueous solution is not less than 32 mg / ml.

[0049] Furthermore, the concentration of the recombinant protein in the aqueous solution is 32 to 75 mg / ml. When the working concentration is greater than or equal to 32 mg / ml, a supramolecular hydrogel with characteristics such as shear thinning effect and injectability will be formed. The formation of this hydrogel does not require any additional artificial interference, such as adjusting pH, adding chemical cross-linking agents, etc.; the working environment can be any aqueous solution, including pure water and various buffer solutions, and the ambient temperature is not limited, such as 0°C-room temperature; at a lower concentration (32 mg / ml), it takes about 5 hours to complete the formation of the hydrogel, and at a high concentration (75 mg / ml), it only takes 30 minutes to complete the gelation. Of course, different recombinant proteins can also be mixed in any proportion to prepare the hydrogel.

[0050] The second object of the present invention is to provide a hydrogel prepared by the above method.

[0051] The third object of the present invention is to provide application of the hydrogel in biological or medical fields.

[0052] Furthermore, the applications include but are not limited to injectable gels, tissue engineering, drug delivery, enzyme immobilization, etc.

[0053] The primers used in the following examples are listed below:

[0054]

[0055] The aqueous solutions involved in the following embodiments include but are not limited to:

[0056] Pure water, and various buffer solutions such as PBS buffer, Tris-HCl buffer, phosphate buffer, etc.

[0057] Example 1 Obtaining the amyloid fibril short peptide Ure2 (1-80)

[0058] Ure2(1-80) can be obtained from the genome clone of Saccharomyces cerevisiae or directly obtained by gene synthesis. In this case, the gene synthesis method was used, and the amino acid sequence of the short peptide is:

[0059] MMNNNGNQVSNLSNALRQVNIGNRNSNTTTDQSNINFEFSTGVNNN NNNNSSSNNNNVQNNNSGRNGSQNNDNENNIKNT.

[0060] The corresponding gene sequence can be of many kinds due to codon preference, but as long as the amino acid sequence encoded in the end is as shown above, it is the target sequence in the present invention.

[0061] Example 2 Fusion of the amyloid fiber short peptide Ure2 (1-80) to the N-terminus of green fluorescent protein (EGFP)

[0062] The amyloid fibril peptide Ure2(1-80) was fused to the N-terminus of green fluorescent protein (EGFP) using primers Pf_Ure2(1-80)(N) / Pr_Ure2(1-80)(N), Pf_Ure2(1-80)-GFP(N) / Pr_Ure2(1-80)-GFP(N) and the Gibson assembly kit (New England Biolabs) and inserted into the NdeI / XhoI of the expression plasmid pET-28a(+), thus constructing the expression plasmid pET28a-Ure2(1-80)-EGFP.

[0063] Example 3: Fusing the short peptide mutant Ure2 (jieduan) with 16 amino acids truncated from the C segment to the N-terminus of green fluorescent protein (EGFP)

[0064] The truncated amyloid fibril peptide Ure2(jieduan) was fused to the N-terminus of green fluorescent protein (EGFP) using primers Pf_Ure2(jieduan)(N) / Pr_Ure2(jieduan)(N), Pf_Ure2(jieduan)-GFP(N) / Pr_Ure2(jieduan)-GFP(N) and the Gibson assembly kit (New England Biolabs) and inserted into the NdeI / XhoI of the expression plasmid pET-28a(+), thus constructing the expression plasmid pET28a-Ure2(jieduan)-EGFP.

[0065] Example 4: Fusing the short peptide mutant Ure2 (tubian) with random mutations of 8 amino acids to the N-terminus of green fluorescent protein (EGFP)

[0066] The amyloid fiber peptide Ure2 (1-80) was randomly mutated by 8 amino acids, accounting for 10% of the total peptide sequence, by gene synthesis. The mutated 8 amino acid sites were randomly selected to prove that the self-assembly peptide can arbitrarily change 10% of its own sequence, but still does not change its self-assembly activity. The mutant amyloid fiber peptide Ure2 (mutation) was fused to the N-terminus of green fluorescent protein (EGFP) using primers Pf_Ure2 (tubian) (N) / Pr_Ure2 (tubian) (N), Pf_Ure2 (tubian)-GFP (N) / Pr_Ure2 (jieduan)-GFP (N) and Gibson assembly kit (New England Biolabs) and inserted into the NdeI / XhoI of the expression plasmid pET-28a (+), thus constructing the expression plasmid pET28a-Ure2 (tubian)-EGFP.

[0067] Example 5 Expression and purification of target protein

[0068] Preparation of culture medium: LB (Luria broth) liquid culture medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0; sterilize with high pressure steam at 121°C for 20 min.

[0069] LB solid medium: add 2% agar powder to LB (Luria broth) liquid medium; sterilize with high pressure steam at 121°C for 20 min.

[0070] Preparation of competent E. coli:

[0071] The expression strain selected in this embodiment is Escherichia coli BL21 (DE3). Pick a single colony in 5mL LB medium and shake culture (37°C, 200rmp) overnight (about 12 hours). Take 500μL of overnight culture and transfer it to 50mL fresh LB medium, shake culture (37°C, 200rmp) until the absorbance reaches 0.35-0.6 at 600nm. Precool 0.1mol / L CaCl2 solution on ice. Pipette 25mL bacterial solution into 50mL EP tube and cool on ice for 10min. Centrifuge (3000g, 5min, 4°C), discard supernatant, add 1.666mL precooled 0.1mol / L CaCl2 solution, gently aspirate up and down with a pipette to mix well, resuspend the cells, and place on ice for 20min. Centrifuge (3000g, 5min, 4℃), discard the supernatant, add 1mL of pre-cooled mixed solution (0.1mol / LCaCl2 mixed with 10% glycerol), pipette and mix to resuspend the cells. Dispense 100μL of cell suspension into 1.5mL EP tubes in each tube and store at -70℃ for future use.

[0072] Transformation of plasmid into Escherichia coli BL21:

[0073] After the competent cells melt, transfer 1ul of plasmid into 100μL competent cells and flick gently to mix. Place on ice for 30min, heat shock in a 42℃ water bath for 90s, and place on ice for 10min. Add 1mL of fresh LB medium to the competent cell EP tube and culture in a shaker (37℃, 200rpm, 1H). Add all E. coli to the kanamycin resistance plate and spread it evenly. Place the transformation plate in a 37℃ incubator and culture it upside down for 12-24H.

[0074] Strain fermentation:

[0075] Prepare the strain activation medium (LB liquid medium), add the engineered bacteria at a ratio of 1% to the strain activation LB medium, and culture on a shaker (37°C, 200 rpm) for 10-12 hours. 600 The initial inoculum size of 0.1 was inoculated into 200 mL of fermentation medium (LB liquid medium), and cultured on a shaker (37°C, 200 rpm) until the absorbance at 600 nm reached 0.5-0.6, then 10 mM IPTG was added, and the fermentation was induced on a shaker (30°C, 200 rpm) for 10-12 hours, and the absorbance at 600 nm was measured to be 2.0-2.5.

[0076] Collecting bacteria:

[0077] After removing the supernatant by high-speed refrigerated centrifuge at 6000 rpm, 10 min, and 4°C, 200 mL of fermentation broth was transferred to 50 mL EP tubes in batches, and the obtained bacteria were placed on a vortex instrument to resuspend the bacteria evenly and then ultrasonically disrupted in an ice bath.

[0078] The ultrasonic instrument parameters were set as follows: power 900 W, probe size 12 mm, amplitude 60%, ultrasonication for 2 s and stop for 2 s, total time 2 min, and ultrasonication for a total of 6 min.

[0079] Protein purification:

[0080] Transfer the cell lysate to a 50mL EP tube, centrifuge at 8000rpm, 10min, 4℃, carefully transfer the supernatant to a clean tube without touching the precipitate below, mix the supernatant with 5mL Ni-NTA agarose purification resin, and incubate on a mixer for 1h. The water and buffer used were filtered with a 0.22um or 0.45um filter membrane before use to reduce the impact of impurities on the filler.

[0081] Wash with 10 column volumes of Stripping Buffer (50mM NaH2PO4, 300mM NaCl, 100mM EDTA, pH8.0), then wash the resin with 10-20 column volumes of deionized water, then wash the resin with 5 column volumes of 100mM NiSO4 (dissolved in deionized water), and finally re-balance with 10 column volumes of 1X PBS. The balanced resin can be used for purification experiments.

[0082] The incubated Ni-NTA agarose purification resin was loaded onto the column for gradient elution. Most of the impurities were eluted with 20 ml of 40 mM imidazole, and then the target protein was eluted with 150 mM imidazole. The protein concentration in the eluate was determined using the modified BCA protein concentration determination kit (C503051), and the eluted protein can be used for SDS-PAGE analysis. The column material was eluted with 5 volumes of ElutionBuffer, and then equilibrated with 5 volumes of Binding / Wash Buffer. Finally, the column material was washed with 5 volumes of ddH2O, and 20% ethanol protective solution was added. It was stored at 2-8°C and cannot be frozen. The collected protein eluate was dialyzed and freeze-dried to finally obtain the target protein, which was stored at 4°C.

[0083] Example 6 Preparation of Nanofiber Self-Assembly Aggregates

[0084] The recombinant protein fused with the above-mentioned self-assembling short peptide is dissolved in pure water or various buffer solutions in a concentration range of 2mg / ml-32mg / ml. The ambient temperature is not limited and can be carried out at 0°C-room temperature. The above-mentioned protein solution is allowed to stand to obtain nanofiber self-assembly aggregates, and the recombinant protein activity of the self-assembly aggregates remains unchanged; the time for self-assembly to be completed is shortened as the concentration of the fusion protein increases. At a low concentration (2mg / ml), it takes about 2 days to stand to complete the entire self-assembly. At a high concentration (32mg / ml), it takes about 5 hours to stand to complete the entire self-assembly.

[0085] Example 7 Preparation of supramolecular hydrogel

[0086] The method for preparing the nanofiber self-assembling hydrogel is as follows: a recombinant protein fused with the above-mentioned self-assembling short peptide is dissolved in pure water or various buffer solutions, the concentration range is greater than 32 mg / ml, the ambient temperature is not limited, and it can be carried out at 0°C-room temperature, and the above-mentioned protein solution is allowed to stand to obtain a nanofiber self-assembling hydrogel, and the activity of the recombinant protein in the hydrogel remains unchanged; the time for self-assembly to be completed is shortened as the concentration of the fusion protein increases. At a lower concentration (32 mg / ml), it takes about 5 hours to stand to complete the generation of the hydrogel, and at a high concentration (75 mg / ml), it only takes 30 minutes to stand to complete the gelation.

[0087] Example 8 Preparation of composite hydrogel

[0088] Different recombinant proteins fused with the above self-assembling short peptides are dissolved in pure water or various buffers, with a concentration range of more than 10 mg / ml. The ambient temperature is not limited and can be carried out at 0°C-room temperature. The above protein solutions are mixed in any proportion and allowed to stand to obtain a nanofiber-like self-assembling composite hydrogel. Different recombinant protein components in the composite hydrogel are evenly mixed and all maintain activity. The time for self-assembly to complete is shortened as the concentration of the fusion protein increases. At a lower concentration (32 mg / ml), it takes about 5 hours to complete the generation of the hydrogel. At a high concentration (75 mg / ml), it only takes 30 minutes to complete the gelation.

[0089] Example 9 Detection of Nanofiber Formation Using Thioflavin T (ThT)

[0090] Thioflavin T (Thioflavin T, ThT) will bind to the formed nanofibers to emit fluorescence, so the target protein aqueous solution is mixed with 20mM Thioflavin T (Thioflavin T, ThT) and the fluorescence value is measured at a fixed wavelength / excitation wavelength of 440 / 530nm. All fluorescence measurements are normalized by dividing the measured fluorescence value by the OD600 of the well. Cell fluorescence and cell density (OD600) are measured by a Cystation 3 imaging reader system (BioTek, Winooski, USA). Three biological replicates were performed and the average value was taken for each sample.

[0091] Example 10 TEM observation of nanofiber formation

[0092] For negative stain TEM, a 10 μL drop of target protein suspension (30 μM) was loaded onto a glow-discharged carbon-coated grid and kept for 1 minute. The extra sample was removed by blotting with filter paper and kept for 1 minute. 2% uranyl acetate was used to stain the sample for 20 seconds. A CM120-FEG (FEI) microscope operated at 100 kV was used to record the micrographs.

[0093] Example 11 Rheological determination of supramolecular hydrogel

[0094] The rheological properties of supramolecular hydrogels were studied using a strain-controlled rheometer (TA Instruments, AR-G2). All measurements were performed on three independently prepared hydrogel samples. The hydrogel samples prepared at different concentrations were placed on a plate. The rheological experiments were performed by placing different concentrations on a plate. The strain-dependent strain scanning mode was then used with an amplitude range of 0.01% to 1000% and a fixed frequency of 1 rad / s or a fixed frequency scanning mode with a strain of 1%, a frequency scanning mode of 0.01 rad / s to 10 rad / s, while the storage modulus (G') and the loss modulus (G") were continuously measured.

[0095] The results of the above examples are as follows:

[0096] Figure 1 Aggregates formed by green fluorescent protein fused with self-assembling short peptide Ure2(1-80) (Ure2(1-80)-EGFP) at low concentration (2 mg / ml), where A represents the control, which is green fluorescent protein (EGFP) without fusion short peptide, and B represents Ure2(1-80)-EGFP;

[0097] Figure 2 Aggregates formed by green fluorescent protein (Ure2(jieduan8)-EGFP) fused with a short peptide mutant Ure2(jieduan8) with 8 amino acids truncated from its C segment at low concentration (2 mg / ml);

[0098] Figure 3 Aggregates formed by green fluorescent protein fused with a short peptide mutant Ure2(jieduan) with 16 amino acids truncated from its C segment (Ure2(jieduan)-EGFP) at low concentration (2 mg / ml);

[0099] Figure 4 Aggregates formed by green fluorescent protein (Ure2(tubian)-EGFP) fused with a short peptide mutant Ure2(tubian) with four random amino acid mutations at low concentrations (2 mg / ml);

[0100] Figure 5 Aggregates formed by green fluorescent protein (Ure2(tubian)-EGFP) fused with a short peptide mutant Ure2(tubian) with random mutations of 8 amino acids at low concentration (2 mg / ml);

[0101] Figure 6 Nanofiber formation efficiency of fluorescent protein fused with different self-assembling peptides. Control is fluorescent protein (EGFP) without self-assembling peptide fusion

[0102] Figure 7 Ure2(1-80)-EGFP fusion protein formed nanofiber structure under transmission electron microscopy;

[0103] Figure 8 Ure2(jieduan)-EGFP fusion protein formed nanofiber structure under transmission electron microscopy;

[0104] Fig. 9 Ure2(tubian)-EGFP fusion protein formed nanofiber structure under transmission electron microscopy;

[0105] Fig.10 Active self-assembled aggregates formed by Ure2(1-80)-EGFP at different concentrations; A: aggregates under white light, B: aggregates under ultraviolet light. Since the aggregates emit fluorescence under ultraviolet light, it shows that the fluorescent protein in the aggregates is an active assembly. As can be seen from the figure, when the concentration is lower than 32 mg / ml, obvious precipitation is visible and no gel is formed, while when the concentration reaches 32 mg / ml, a hydrogel can be formed.

[0106] Fig.11 Thioflavin T (ThT) was used to detect the formation of nanofibers of Ure2(1-80)-EGFP at different concentrations.

[0107] Fig.12Nanohydrogels formed by Ure2(1-80)-EGFP(A) and Ure2(1-80)-mCherry(B) at low concentrations (32mg / ml). A: Green fluorescent protein fused with the self-assembling short peptide Ure2(1-80), B: Red fluorescent protein fused with the self-assembling short peptide Ure2(1-80)

[0108] Fig.13 Supramolecular composite hydrogel formed by Ure2(1-80)-EGFP and Ure2(1-80)-mCherry in equal proportions at high concentration (75 mg / ml).

[0109] Fig.14 Supramolecular composite hydrogels formed by Ure2(1-80)-EGFP and Ure2(1-80)-mCherry at low concentrations (32 mg / ml) and in different ratios.

[0110] Fig.15 Rheological properties of supramolecular hydrogels formed with different concentrations of Ure2(1-80)-EGFP. The rheological properties of supramolecular hydrogels were measured using a strain-controlled rheometer (TA Instruments, AR-G2) in amplitude (A) and frequency (B) scanning modes.

[0111] Fig.16 The supramolecular hydrogel formed by the green fluorescent protein (Ure2(jieduan8)-EGFP) fused with the short peptide mutant Ure2(jieduan8) with 8 amino acids truncated in the C segment at a low concentration (32mg / ml);

[0112] Fig.17 The supramolecular hydrogel formed by the green fluorescent protein (Ure2(jieduan)-EGFP) fused with the short peptide mutant Ure2(jieduan) with 16 amino acids truncated in the C segment at high concentration (75mg / ml);

[0113] Fig.18 Supramolecular hydrogel formed by fusion of green fluorescent protein (Ure2(tubian)-EGFP) with a short peptide mutant Ure2(tubian) with four random amino acid mutations at low concentration (32 mg / ml);

[0114] Fig.19 Supramolecular hydrogel formed at high concentration (75 mg / ml) of green fluorescent protein (Ure2(tubian)-EGFP) fused with a short peptide mutant Ure2(tubian) with 8 amino acids randomly mutated.

[0115] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for self-assembling recombinant proteins into supramolecular colloids, characterized in that: The following steps are involved: The target protein is fused with an amyloid short peptide or its mutant, and then one or more recombinant proteins are placed in an aqueous solution for self-assembly; Among them, the amino acid sequence of the amyloid short peptide is: MMNNNGNQVSNLSNALRQVNIGNRNSNTTTDQSNINFEFSTGVNNNNNSSSSNNNNVQNNNSGRNGSQNNDNENNIKNT, The amyloid peptide mutant is any of the following: (a) The amyloid peptide is truncated from the C-terminus to any of the 1st to 16th amino acids; (b) The amyloid peptide undergoes no more than 10% amino acid substitution; The concentration of the recombinant protein in the aqueous solution is above 32 mg / ml.

2. The method according to claim 1, characterized in that: The amyloid peptide or its mutant is fused to the N-terminus or C-terminus of any protein.

3. The method according to claim 1, characterized in that: The recombinant protein is placed in an aqueous solution and allowed to stand to obtain a hydrogel.

4. The method according to claim 3, characterized in that: Let stand for 30min~5h.

5. The hydrogel prepared by the method according to any one of claims 1 to 4.

6. Use of the hydrogel according to claim 5 in the biological or medical fields.

7. The use according to claim 6, characterized in that: The applications are injectable gels, tissue engineering, drug delivery or enzyme immobilization.

Images