Dual-compartment protein hydrogels with controllable isothiocyanate generation and release and applications thereof

Abstract

This invention relates to the field of biotechnology, and more particularly to a double-compartment protein hydrogel with controllable isothiocyanate generation and release, and its applications. Using shrimp tropomyosin and shrimp ferritin as raw materials, this invention constructs a hydrogel capable of compartmentalizing glucosinolates and myrosinase; the hydrogel effectively controls the generation and release of isothiocyanates and exhibits excellent inhibitory effects on microbial growth.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a dual-compartment protein hydrogel with controllable isothiocyanate generation and release and its applications. Background Technology

[0002] Many natural products in plants not only contribute to their unique flavor but also possess potent anti-cancer, antibacterial, and antiviral activities, thus playing a crucial role in food processing and pharmaceutical development. For example, in cruciferous plants, glucosinolates located at different positions, under the action of myrosinase, generate isothiocyanates, compounds with a pungent and spicy taste, which are the main source of black mustard's spiciness. Furthermore, this substance also possesses resistance to pests and diseases and has antiseptic and antibacterial properties. However, isothiocyanates often face instability issues, readily degrading under light, oxygen, and high temperatures, severely limiting their applications in food and medicine. To achieve efficient utilization of isothiocyanates, it is urgent to address their high volatility and degradation rates. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide a dual-compartment protein hydrogel with controllable isothiocyanate generation and release and its application.

[0004] This invention provides a TSF fusion protein, which comprises, from the N-terminus to the C-terminus, a shrimp tropomyosin subunit, a linker peptide, and a shrimp ferritin subunit;

[0005] The amino acid sequence of the shrimp tropomyosin subunit is shown in SEQ ID NO:1;

[0006] The amino acid sequence of the shrimp ferritin subunit is shown in SEQ ID NO:3;

[0007] The linker peptide is (GGGGS)n; n is an integer from 1 to 10. Specifically, in this invention, n is optimal when it is 3.

[0008] In the construction of the TSF fusion protein of this invention, the length of shrimp tropomyosin, the amino acid composition of shrimp ferritin subunits, and the linker peptide sequence were optimized. The results showed that the TSF fusion protein obtained here had the best encapsulation rate of glucosinolate substrate, the best controlled release effect, and the best gelation properties when used for the preparation of double-compartment hydrogels.

[0009] The TSF fusion protein of the present invention may further include a purification tag in its N segment, including but not limited to a His tag.

[0010] Furthermore, the His tag is (H)n, where n is an integer from 1 to 20, and in this invention, n is 6;

[0011] The present invention provides a nucleic acid encoding the TSF fusion protein.

[0012] Furthermore, the nucleotide sequence of the nucleic acid encoding the TSF fusion protein is shown in SEQ ID NO:6.

[0013] The present invention optimizes the nucleic acid encoding the TSF fusion protein, and the results show that the codon-optimized nucleic acid has better soluble expression.

[0014] This invention provides a hydrogel in which the encapsulation product of the TSF fusion protein described in this invention, after encapsulating glucosinolates, is combined with black myrosinase in Zn 2+ It is prepared by mixing under the condition that it exists.

[0015] In the hydrogel of the present invention, the molar ratio of the TSF fusion protein to glucosinolate during encapsulation is 1:(50~500); wherein, 1:200 is optimal.

[0016] The Zn 2+ The concentration ranges from 2 mg / mL to 8 mg / mL.

[0017] The conditions for embedding glucosinolates in the TSF fusion protein are ultrasonic treatment for 30 to 120 minutes, with 90 minutes being the optimal time in this invention.

[0018] The concentration of the black myrosinase is 8 mM to 12 mM, and in a specific embodiment of the present invention it is 10 mM;

[0019] The concentration of TSF fusion protein in the embedded product is 28 mg / mL to 32 mg / mL, and in a specific embodiment of the present invention it is 30 mg / mL.

[0020] The host cell expressing the TSF fusion protein was BL21(DE3), the induction temperature was 20°C, and the nucleotide sequence of the nucleic acid encoding the TSF fusion protein is shown in SEQ ID NO:6.

[0021] This invention provides the use of the TSF fusion protein and / or the hydrogel in the preparation of anticancer, antibacterial and / or antiviral drugs.

[0022] This invention provides a two-compartment protein hydrogel for controllable isothiocyanate generation and release, and its applications. First, protein engineering is used to fuse and express fibrous shrimp tropomyosin and cage-like shrimp ferritin, creating two distinct compartments on a single material. The first compartment of this two-compartment protein encapsulates glucosinolates, and the second compartment encapsulates myrosinase. Finally, metal ion cross-linking is used to form the second compartment, thus preparing the hydrogel. Through these steps, a two-compartment protein is prepared to separate the encapsulated enzyme and substrate, thereby controlling the generation and release of the product isothiocyanate. This invention provides a new approach for developing high-retention technologies for unstable flavor substances and expands the application of such active compounds in inhibiting microbial growth. The preparation method of this invention is simple, low-cost, and exhibits significant functional activity, aligning with the goals of sustainable socio-economic development.

[0023] This invention uses shrimp tropomyosin and shrimp ferritin as raw materials to construct a hydrogel that can compartmentally separate glucosinolates and myrosinase. The hydrogel effectively controls the generation and release of isothiocyanates and has a good inhibitory effect on microbial growth. Attached Figure Description

[0024] Figure 1 A schematic diagram illustrating the design of a dual-compartment protein;

[0025] Figure 2 Transmission electron microscopy characterization of TSF double-compartment proteins;

[0026] Figure 3 The preparation process of a double-compartment protein hydrogel with controllable isothiocyanate generation and release is shown.

[0027] Figure 4 Transmission electron microscopy characterization of TSF-encapsulated glucosinolates;

[0028] Figure 5 The transmission electron microscope and images of the prepared double-compartment protein hydrogel are shown.

[0029] Figure 6 The results of scanning electron microscopy are shown for the prepared double-compartment protein hydrogel.

[0030] Figure 7 Show the standard curve of glucosinolates;

[0031] Figure 8 This shows the encapsulation efficiency and drug loading of TSF protein-encapsulated glucosinolates in Example 1;

[0032] Figure 9 This shows the standard curve for isothiocyanates.

[0033] Figure 10The formation of isothiocyanate in the hydrogels of Example 1 and Comparative Example 4 within 24 hours is shown.

[0034] Figure 11 This demonstrates the effect of a double-compartment protein hydrogel containing glucosinolates and myrosinase on inhibiting microbial growth.

[0035] Figure 12 Nanostructures of fusion proteins formed from tropomyosin of different lengths are shown;

[0036] Figure 13 The gelation of fusion proteins formed from tropomyosin of different lengths is shown.

[0037] Figure 14 This example demonstrates the encapsulation efficiency and drug loading of fusion proteins containing glucosinolates prepared with different ferritin primary structures in Example 3.

[0038] Figure 15 Gel sputtering images showing the expression of fusion proteins prepared with different linker peptides in Example 4;

[0039] Figure 16 This example shows the encapsulation efficiency and drug loading of TSF protein encapsulating glucosinolates under different molar ratios during ultrasonic encapsulation in Example 5.

[0040] Figure 17 This shows the encapsulation rate and drug loading of TSF protein-encapsulated glucosinolates under different ultrasound times in Example 6.

[0041] Figure 18 Transmission electron microscopy characterization of the fusion protein TSF produced by Rosetta(DE3) cells;

[0042] Figure 19 Gel sputtering images showing the TSF yield of the fusion protein during induction at 16℃ and 37℃. Detailed Implementation

[0043] This invention provides a dual-compartment protein hydrogel with controllable isothiocyanate generation and release, and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0044] The amino acid sequence of the shrimp tropomyosin subunit: DAIKKKMQAMKLEKDNAMDRADTLEQQNKEANNRAEKAEDEVNKLQKKMQQLENDLDQVQESLLTANNQLEEKDKALSNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAADESERARKCLENRANMEDDRVGILEAQLAQAKHIAEEADKKYEELARKSVMLENDLERAEERAEAAEGKIVELEEELRVVGNNLKSLEVSEEKANQREEAYKEQIKTLTNKLKAAEARAEFAERSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSELSGY (SEQ ID NO:1);

[0045] The amino acid sequence of the ferritin subunit of wild-type shrimp: ASQVRQNYHEDCEASINKQINMELYASYVYLSMAYYFERDDVALPGFAKFFKESSDEEREHAQTFMKYQNKRGGRIVLQQIAAPSMQEWGTGLEALQAALDLEKQVNQSLLELHSTASGNNDPHLTKLLEDEYLEEQVDSIKKIGDMITKLKRAGPTGLGEYMFDKELN (SEQ ID NO:2);

[0046] The amino acid sequence of the mutated shrimp ferritin subunit is: ASQVRQNYHEDCEASINKQINMELYASYVYLSMAYYFERDDVALPGFAKFFKKSSDKERKHAQTFMKYQNKRGGRIVLQQIAAPSMREWGTGLEALQAALDLEKQVNQSLLELHSTASGNNDSHLTSLLSSEYLKKQVDSIKKIGDMITKLKRAGPTGLGEYSFDKSLN (SEQ ID NO:3);

[0047] The amino acid sequence of the mutant TSF fusion protein (mutation positions are indicated in bold): MHHHHHHDAIKKKMQAMKLEKDNAMDRADTLEQQNKEANNRAEKAEDEVNKLQKKMQQLENDLDQVQESLLTANNQLEEKDKALSNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAADESERARKCLENRANMEDDRVGILEAQLAQAKHIAEEADKKYEELARKSVMLENDLERAEERAEAAEGKIVELEEELRVVGNNLKSLEVSEEKANQREE AYKEQIKTLTNKLKAAEARAEFAERSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSELSGYGGGGSGGGGSGGGGSASQVRQNYHEDCEASINKQINMELYASYVYLSMAYYFERDDVALPG FAKFFKKSSDKERKHAQTFMKYQNKRGGRIVLQQIAAPSMREWGTGLEALQAALDLEKQVNQSLLELHSTASGNNDSHLTSLLSSEYLKKQVDSIKKIGDMITKLKRAGPTGLGEYSFDKSLN (SEQ ID NO:4);

[0048] Linker peptide 1: GGGGSGGGGSGGGGS (SEQ ID NO:5);

[0049] The nucleotide sequence of the codon-optimized mutant TSF fusion protein: (SEQ ID NO:6);

[0050]

[0051] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0052] Example 1: Preparation of a two-compartment protein hydrogel with controllable isothiocyanate generation and release

[0053] I. Expression of Fusion Proteins

[0054] (1) A double-compartment fusion protein TSF was designed using protein engineering. Specifically, the entire sequence consists of three domains, from the N-terminus to the C-terminus: histidine-shrimp tropomyosin subunit (SEQ ID NO:1)-linker (linking peptide 1, SEQ ID NO:5)-shrimp ferritin subunit (SEQ ID NO:3). The first domain is the shrimp ferritin subunit, the second domain is the shrimp tropomyosin subunit, and the third domain is a motif consisting of 6 histidine residues. The first and second domains are connected by a flexible linker. The cavity structure of ferritin is the designed first compartment, and the second compartment is the fibrous structure of tropomyosin connected by Zn. 2+ Induced formation ( Figure 1 ).

[0055] The coding gene for the entire fusion protein TSF was synthesized by Qingke Biotechnology Co., Ltd. after codon optimization. The nucleotide sequence is shown in SEQ ID NO:6. The nucleotide sequence was cloned into the pET-32a vector and transformed into E. coli BL21(DE3) cells. Correct positive clones were selected and seeded into LB medium. When the OD 600 reached 0.8, 0.2 mM IPTG was added to induce protein expression, and the cells were cultured at 20°C for 16-20 hours. After centrifugation at 5000 g for 10 minutes, the cells were collected, resuspended in buffer A (25 mM Tris-HCl, 100 mM NaCl, pH 8.0), and sonicated (270 W, 20 minutes). The lysate was heat-treated (70°C, 10 minutes), salted out with 45% ammonium sulfate, and the precipitate was resuspended and dialyzed three times for 8 hours each time. The dialyzed protein was purified using a Ni-NTA affinity chromatography column, eluted with 200 mM imidazole, and then dialyzed back into buffer A. The purity and concentration of the protein were determined by SDS-PAGE and BCA methods. The expression level of the TSF fusion protein was approximately 50 mg / L, and its morphology was characterized by transmission electron microscopy (TEM). The results showed... Figure 2 middle.

[0056] II. Preparation of a double-compartment protein hydrogel with controllable isothiocyanate generation and release

[0057] 1. Encapsulation of glucosinolates

[0058] The process is as follows Figure 3 As shown; specifically, glucosinolates are embedded into the first compartment of the fusion protein TSF using ultrasound. The specific steps are as follows:

[0059] A mixture of TSF protein (fusion protein TSF):glucosinolate at a concentration ratio of 1:200 (i.e., 1 µM TSF protein and 0.2 mM glucosinolate) was sonicated at 100 W for 90 minutes to obtain the first embedded structure formed by glucosinolate and TSF protein, which was then characterized by TEM. Figure 4 As shown, in the material loaded with glucosinolates via ultrasound, the hollow structure of the first compartment composed of ferritin was embedded by glucosinolates, and the hollow structure disappeared.

[0060] 2. Encapsulation of black myrosinase

[0061] The concentration of TSF protein in the first embedded structure was adjusted to 30 mg / mL. 10 mM myrosinase was mixed with the first embedded structure and stirred for 10 minutes. Then, 5 mg / mL Zn was added. 2+ The formation of a second compartment was induced, resulting in a double-compartment protein hydrogel (referred to as hydrogel) containing glucosinolates and myrosinase. The cross-linked network structure could be observed under TEM. Figure 5 (a) In the inverted bottle experiment, the hydrogel can be seen to remain stably at the bottom of the bottle and will not flow down when the bottle is inverted. Figure 5 b). Scanning electron microscopy (SEM) reveals a uniform network structure ( Figure 6 At physiological pH, it can achieve slow release of glucosinolates, which, upon contact with myrosinase, will generate isothiocyanate in situ and exert its activity.

[0062] III. Performance Testing

[0063] 1. Encapsulation efficiency of glucosinolates

[0064] The TSF fusion protein undergoes amino acid mutations in its ferritin region. Proline (Pro, P), lysine (Lys, K), glutamic acid (Glu, E), aspartic acid (Asp, D), and methionine (Met, M) at the triple and quadruple axis channels of the ferritin are mutated to serine (Ser, S). Specifically, positions 428, 432, 435, 436, 468, and 472 of the fusion protein are mutated to S; and glutamic acid (Glu, E) on the inner surface of the ferritin is mutated to lysine (Lys, K) to reduce the negative potential of the inner surface and decrease electrostatic repulsion to negatively charged substrates. Specifically, positions 358, 362, 365, 440, and 441 of the fusion protein are mutated to K. The amino acid sequence is shown in SEQ ID NO:3. The mutated gene sequence was expressed, purified, and encapsulated with glucosinolates as described above. The encapsulation efficiency and drug loading of glucosinolates were determined according to the following method.

[0065] a. Determination of glucosinolate loading and encapsulation efficiency:

[0066] The content of glucosinolates was determined by high performance liquid chromatography (HPLC), and a quantitative analytical system was established. The chromatographic conditions were set as follows: C18 reversed-phase column, mobile phase of methanol:water (1:9, v / v), detection wavelength of 229 nm, and flow rate of 1 mL / min.

[0067] b. Construction of Standard Curve

[0068] With glucosinolate concentration (0 µM~800 µM) as the abscissa (X) and the corresponding peak area as the ordinate (Y), a linear regression equation was established: Y = 6909X - 640.7 Figure 7 ), R 2 =0.9980, this equation is used for the accurate calculation of drug concentration in subsequent experiments.

[0069] c. Calculation of drug loading index

[0070] Total protein carriers (W) carrier ): Total mass of protein added to the system.

[0071] Total dosage (W) total ): Record the molar mass of glucosinolate initially added during the ultrasonic embedding process.

[0072] Drug loading concentration (C) encapsulatedThe ultrasonically embedded system (first embedded structure) was dialyzed three times for 8 hours each time to remove free glucosinolates. An organic solvent (methanol or acetonitrile) was then added to fully disrupt the protein structure and release the embedded glucosinolates. After adjusting the volume, the mixture was filtered through a 0.22 µm filter membrane. The peak area was measured by HPLC and substituted into the standard curve. The concentration of embedded glucosinolates in the system was calculated using the corresponding dilution factor.

[0073] Encapsulation rate EE (%) = C encapsulated *V / W total ×100%, where V is the total volume of the system;

[0074] Drug loading = C encapsulated *V / W carrier V is the total volume of the system;

[0075] The encapsulation efficiency of glucosinolates was determined to be 32.6%, with a drug loading of 50.6 µmol / g. Figure 8 As shown.

[0076] 2. Formation of isothiocyanates

[0077] The isothiocyanate release behavior of hydrogels obtained by encapsulation with glucosinolates and myrosinase was determined over 12–24 hours. The detection procedure for isothiocyanate release behavior is as follows:

[0078] Mix 50 µL of the sample to be tested (hydrogel) with 450 µL of pH 8.5 phosphate buffer, add 0.5 mL of 4 mM 1,2-benzenedithiophenol (dissolved in methanol), react at 65°C for 1-2 hours, and measure the absorption peak of the reaction product at 365 nm.

[0079] Quantification was performed using a standard curve established with isothiocyanate standards. The isothiocyanate concentration (0 µM~500 µM) was plotted on the x-axis (X), and the corresponding absorbance on the y-axis (Y). A linear regression equation was established: Y = 0.0009803X + 0.06458 ( Figure 9 ), R 2 =0.9949, this equation is used for subsequent precise quantification of isothiocyanate amounts.

[0080] It was determined that isothiocyanates could be continuously generated within 24 hours, as shown in the following results. Figure 10 As shown.

[0081] 3. Antibacterial effect of double-compartment protein hydrogel

[0082] The antibacterial effect of the double-compartment protein hydrogel was determined using the plate count method. 1 mL of a suspension of *E. coli* and *S. aureus* (10 mL) was used.4 The mixture (CFU / mL) was co-incubated with 500 µL hydrogels of different components for 2 hours. Then, a certain amount of the mixture (100 µL) was plated after incubation and counted after 24 hours of incubation.

[0083] The results are as follows Figure 11 As shown in the figure. Control group: bacterial count showed logarithmic growth, with dense colonies. TSF hydrogel group: exhibited extremely strong bactericidal ability. Quantitative results showed that this group had an inhibition rate of over 80% against E. coli and S. aureus.

[0084] Example 2: Optimization of Shrimp Tromyosin Sequence

[0085] I. Design of Shrimp Tromyosin Proteins of Different Lengths

[0086] To investigate the effect of tropomyosin (TM) length on gel properties, three length gradients were designed while maintaining the integrity of its linear sequence, and these gradients were recombinantly expressed with wild-type shrimp ferritin.

[0087] The amino acid sequence of shrimp tropomyosin was not modified, but its length was optimized. Since tropomyosin is a linear protein, its length is crucial for gel formation.

[0088] The tropomyosin sequence is shown in SEQ ID NO:1, consisting of 283 amino acids.

[0089] TM-150 (the length of the first 150 amino acids of tropomyosin retained): DAIKKKMQAMKLEKDNAMDRADTLEQQNKEANNRAEKAEDEVNKLQKKMQQLENDLDQVQESLLTANNQLEEKDKALSNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAADESERARKCLENRANMEDDRVGILEAQLAQA (SEQ ID NO:7);

[0090] The fusion sequence of TM-150 with wild-type shrimp ferritin and histidine tags is: MHHHHHHDAIKKKMQAMKLEKDNAMDRADTLEQQNKEANNRAEKAEDEVNKLQKKMQQLENDLDQVQESLLTANNQLEEKDKALSNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAADESERARKCLENRANMEDDRVGILEAQLAQAGGGGSGGGGSGGGSASQVRQNYHEDCEASINKQINMELYASYVYLSMAYYFERDDVALPGFAKFFKESSDEEREHAQTFMKYQNKRGGRIVLQQIAAPSMQEWGTGLEALQAALDLEKQVNQSLLELHSTASGNNDPHLTKLLEDEYLEEQVDSIKKIGDMITKLKRAGPTGLGEYMFDKELN (SEQ ID NO:8);

[0091] TM-210 (Troposomalin retaining the length of the first 210 amino acids): DAIKKKMQAMKLEKDNAMDRADTLEQQNKEANNRAEKAEDEVNKLQKKMQQLENDLDQVQESLLTANNQLEEKDKALSNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAADESERARKCLENRANMEDDRVGILEAQLAQAKHIAEEADKKYEELARKSVMLENDLERAEERAEAAEGKIVELEEELRVVGNNLKSLEVSE (SEQ ID NO:9);

[0092] The fusion sequence of TM-210 with wild-type shrimp ferritin and histidine tags is: MHHHHHHDAIKKKMQAMKLEKDNAMDRADTLEQQNKEANNRAEKAEDEVNKLQKKMQQLENDLDQVQESLLTANNQLEEKDKALSNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAADESERARKCLENRANMEDDRVGILEAQLAQAKHIAEEADKKYEELARKSVMLENDLERAEER AEAAEGKIVELEEELRVVGNNLKSLEVSEGGGGSGGGGSGGGGSASQVRQNYHEDCEASINKQINMELYASYVYLSMAYYFERDDVALPGFAKFFKESSDEEREHAQT FMKYQNKRGGRIVLQQIAAPSMQEWGTGLEALQAALDLEKQVNQSLLELHSTASGNNDPHLTKLLEDEYLEEQVDSIKKIGDMITKLKRAGPTGLGEYMFDKELN (SEQ ID NO:10);

[0093] TM-283 is SEQ ID NO:1;

[0094] The fusion sequence of TM-283 with wild-type shrimp ferritin and histidine tags is: MHHHHHHDAIKKKMQAMKLEKDNAMDRADTLEQQNKEANNRAEKAEDEVNKLQKKMQQLENDLDQVQESLLTANNQLEEKDKALSNAEGEVAALNRRIQLLEEDLERSEERLNTATTKLAEASQAADESERARKCLENRANMEDDRVGILEAQLAQAKHIAEEADKKYEELARKSVMLENDLERAEERAEAAEGKIVELEEELRVVGNNLKSLEVSEEKANQREEAYKEQIKTLTNKLK AAEARAEFAERSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSELSGYGGGGSGGGGSGGGGSASQVRQNYHEDCEASINKQINMELYASYVYLSMAYYFERDDVALPGFAKFFKE SSDEEREHAQTFMKYQNKRGGRIVLQQIAAPSMQEWGTGLEALQAALDLEKQVNQSLLELHSTASGNNDPHLTKLLEDEYLEEQVDSIKKIGDMITKLKRAGPTGLGEYMFDKELN (SEQ ID NO:11);

[0095] II. Performance Testing of Hydrogels Obtained from Preparation of Shrimp Tromyosin of Different Lengths

[0096] 1. Structural characterization and gelation performance analysis:

[0097] All three lengths of tropomyosin were successfully expressed after fusion with ferritin and histidine tags without affecting ferritin assembly, and all were 24-mers.

[0098] 2. Nanostructure observation

[0099] Under an electron microscope, TM-150 only showed typical ferritin spherical particles, with no obvious peripheral linear structure. Figure 12 (a) In electron microscopy, TM-210 shows short and indistinct linear protrusions distributed around spherical particles. Figure 12 (b) Only in TM-283, which retains the full length of tropomyosin, is a clear "sphere-line" complex structure presented, with distinct fibrous TM chains attached around the spherical core, confirming the advantage of full-length TM in maintaining spatial conformation. Figure 12 (c in the text)

[0100] 3. Analysis of Zinc Ion-Induced Gel Properties

[0101] Add 5 mg / mL Zn to three protein solutions at 30 mg / mL. 2+ The gelling ability of the TM-150 system was examined. The system remained in a flowing liquid state and could not form a network structure. Figure 13 (a) TM-210 formed a turbid liquid, indicating that partial aggregation had occurred, but was insufficient to support the three-dimensional gel network. Figure 13 (b) The TM-283 system can successfully construct stable hydrogel structures that are stable in vials and do not flow when inverted. Figure 13 (c in the text)

[0102] Conclusion: The sequence length of tropomyosin is a key factor in inducing gelation. Only when TM maintains its full-length 283aa sequence can it provide sufficient interaction sites and spatial length to form a physical / chemical cross-linking network sufficient to support the system.

[0103] Example 3: Effect of ferritin primary structure on glucosinolate encapsulation amount

[0104] 1. The ferritin portion uses wild-type ferritin.

[0105] The fusion protein, shrimp ferritin subunit, is wild-type. The specific composition of the fusion protein is: histidine-shrimp tropomyosin subunit (SEQ ID NO:1)-linker (linking peptide 1, SEQ ID NO:5)-shrimp ferritin subunit (SEQ ID NO:2).

[0106] The fusion protein gene sequence was codon optimized and then expressed, purified, and encapsulated with glucosinolates according to the protocol in Example 1. The encapsulation status of glucosinolates was then determined.

[0107] The final encapsulation efficiency of the wild-type sequence for glucosinolates was approximately 18%, with a drug loading of 28 µmol / g. Figure 14 ).

[0108] This indicates that the strong negative potential on the inner surface of wild-type ferritin and the hydrophobic barrier in the channel region cause significant electrostatic repulsion and entry hindrance to negatively charged glucosinolate substrates, resulting in relatively limited overall encapsulation efficiency.

[0109] 2. Hydrophilic mutation in the ferritin fourfold axis channel region - Mutation 2

[0110] The TSF fusion protein (fusion protein TSF) has undergone amino acid mutations in the channel region of its shrimp ferritin subunit. Specifically, proline (Pro, P), lysine (Lys, K), glutamic acid (Glu, E), aspartic acid (Asp, D), and methionine (Met, M) in the channel are mutated to serine (Ser, S). This is achieved by mutating positions 428, 432, 435, 436, 468, and 472 of the fusion protein to S, thereby increasing the hydrophilicity of the channel. The linker peptide is linker peptide 1.

[0111] After codon optimization, the mutated gene sequence was expressed, purified, and encapsulated with glucosinolates according to Example 1, and the encapsulation status of glucosinolates was measured.

[0112] Results: Compared with the wild-type sequence, the fusion protein encapsulation efficiency of this mutant shrimp ferritin subunit was significantly improved, with a final encapsulation efficiency (90 minutes) of approximately 26% and a drug loading of 40.5 µmol / g. Figure 14 This indicates that the increased hydrophilicity of the channel region facilitates the more rapid entry of negatively charged glucosinolates into the ferritin cavity, overcoming the hydrophobic barrier of the channel region.

[0113] 3. Mutation that reduces the negative surface potential of ferritin (E to K) - Mutation 3

[0114] The fusion protein TSF has an inner surface amino acid mutation in its ferritin portion, where glutamic acid (Glu, E) is mutated to lysine (Lys, K). Specifically, E at positions 358, 362, 365, 440, and 441 of the TSF fusion protein is mutated to K.

[0115] The mutated gene sequence was codon-optimized and then expressed and purified according to the optimized scheme of Example 1. Expression, purification, and glucosinolate encapsulation were performed according to the method of Example 1, and the glucosinolate encapsulation rate and drug loading were determined.

[0116] Results: Compared with the wild type, the inner surface negative potential-reduced mutant significantly increased the encapsulation efficiency of glucosinolates to 29.8%, with a drug loading of 46.4 µmol / g. Figure 14 This indicates that the decrease in the negative potential of the inner surface weakens the electrostatic repulsion of the negatively charged substrate, allowing more substrate to be encapsulated.

[0117] Example 4 Optimization of Linker

[0118] 1. Optimization of connecting peptides

[0119] The fusion protein TSF was obtained by directly connecting shrimp tropomyosin and shrimp ferritin without using a linker peptide. The fusion gene was cloned into the pET-32a vector and transformed into E.coli BL21(DE3) competent cells. The target protein was expressed, purified, and subjected to gel induction experiments.

[0120] Results: The protein mainly existed in the form of inclusion bodies, with extremely low yields of soluble protein. No obvious cage-like or fibrous structures were observed under electron microscopy, indicating that the two protein domains interfered with each other and failed to fold correctly.

[0121] 2. Optimization of linker peptides

[0122] Based on the above results, this invention uses different linking peptides to investigate the expression, solubility, and correct folding of the fusion protein;

[0123] Specifically, different linker peptides are used to link shrimp tropomyosin and shrimp ferritin to obtain fusion proteins:

[0124] Linker peptide 2: GGGGS (flexible linker), SEQ ID NO:12;

[0125] Linker peptide 3: GGGGSGGGGS (flexible linker), SEQ ID NO:13;

[0126] Linker peptide 4: EAAAK (rigid linker), SEQ ID NO:14;

[0127] The fusion protein gene was cloned into the pET-32a vector and transformed into E.coli BL21(DE3) competent cells. The target protein was expressed, purified, and subjected to gel induction experiments.

[0128] result:

[0129] Using linker peptide 2, some soluble protein was obtained, but the yield was significantly lower than in Example 1. Electron microscopy revealed some ferritin cage-like structures, but tropomyosin fibrillation was not obvious, or aggregation occurred.

[0130] Using linker peptide 3, some soluble protein was obtained, but the yield was lower than in Example 1. Partial ferritin cage structures were visible under electron microscopy.

[0131] Using linker peptide 4, a small amount of soluble protein was obtained, but the yield was lower than in Example 1. Cage-like and fibrous structures were barely visible under electron microscopy, indicating that the rigid linker could not satisfy the separation between the two protein domains.

[0132] Gel sprites showing the expression of the fusion protein TSF using different linking peptides are shown below. Figure 15As shown, using the linker peptide 1 of this invention is the optimal solution for achieving efficient soluble expression of TSF protein and correct formation of the double-compartment structure. Other linker peptides are not conducive to the correct folding, soluble expression, and double-compartment structure formation of the fusion protein TSF (shrimp tropomyosin-shrimp ferritin).

[0133] Table 1. Expression and assembly of TSF fusion proteins using different linker peptides

[0134]

[0135] Example 5: Change in the mass ratio of TSF protein to glucosinolates to 1:200

[0136] The procedure was the same as in Example 1, except that the concentration ratios of the fusion protein TSF to glucosinolates were set to 1:50, 1:100, 1:300, and 1:500, respectively. After encapsulation, free glucosinolates were separated by ultrafiltration centrifugation, and the encapsulation efficiency was determined by high-performance liquid chromatography.

[0137] result( Figure 16 ):

[0138] When the concentration ratio of fusion protein TSF to glucosinolate was 1:50, the encapsulation efficiency was approximately 78%, the total drug loading was 30.3 µmol / g, and the amount of glucosinolate loaded per unit protein was significantly lower than that in Example 1.

[0139] When the concentration ratio of the fusion protein TSF to glucosinolate was 1:100, the encapsulation efficiency and total drug loading were lower than those in Example 1, at 59.7% and 45.2 µmol / g, respectively.

[0140] When the concentration ratio of the fusion protein TSF to glucosinolate was 1:300, the encapsulation efficiency and total drug loading were 23.8% and 53.7 µmol / g, respectively.

[0141] When the concentration ratio of the fusion protein TSF to glucosinolate was 1:500, the encapsulation efficiency decreased to 14.1%, and the drug loading was 54.5 µmol / g.

[0142] Although the encapsulation efficiency was approximately 78% and 59.7% when the concentration ratio of fusion protein TSF to glucosinolate was 1:50 and 1:100, respectively, which was higher than that of Example 1, this was due to the lower amount of glucosinolate added; the actual drug loading was lower than that of Example 1. When the concentration ratio of fusion protein TSF to glucosinolate was 1:300 and 1:500, the drug loading was close to that of Example 1, but the encapsulation efficiency decreased significantly, indicating that the cavity encapsulation had reached saturation and excess substrate could not be effectively loaded. In summary, Example 1 achieved a balance between high drug loading and relatively high encapsulation efficiency.

[0143] Example 6 Optimization of Ultrasound Time

[0144] The encapsulation of glucosinolates was the same as in Example 1, except that the ultrasonic treatment time was set to 30 minutes, 60 minutes and 120 minutes respectively. After encapsulation, free glucosinolates were separated by ultrafiltration centrifugation and the encapsulation rate was determined by high performance liquid chromatography.

[0145] result( Figure 17 ):

[0146] The embedding rate was 13.4% and the drug loading was 20.8 µmol / g after ultrasonic treatment for 30 minutes, which was significantly lower than that in Example 1.

[0147] The embedding rate was 18.7% and the drug loading was 31.0 µmol / g after ultrasonic treatment for 60 minutes, which was significantly lower than that in Example 1.

[0148] The encapsulation rate was 24.9% and the drug loading was 38.7 µmol / g after 120 minutes of sonication. This is significantly lower than that after 90 minutes, suggesting that prolonged sonication may have caused some protein structures to loosen or cavities to be damaged, resulting in leakage of contents.

[0149] Table 2. Optimization results of embedding process

[0150]

[0151] Comparative Example 1: The gene encoding the fusion protein TSF was not codon-optimized.

[0152] The fusion protein TSF, from N-terminus to C-terminus, consists of histidine-shrimp tropomyosin subunit (SEQ ID NO:1)-linker (linker peptide 1, SEQ ID NO:5)-shrimp ferritin subunit (SEQ ID NO:3). The gene sequence encoding the TSF protein was not optimized for whole-genome synthesis based on the codon usage frequency table of E. coli (derived from shrimp cDNA).

[0153] Protein expression and purification were performed as described in Example 1. After purification, protein concentration was determined using the BCA method, and the yield of soluble protein per liter of bacterial culture was calculated.

[0154] Results: Some soluble protein was obtained, with a content of 7 mg / L, which was significantly lower than that in Example 1 (50 mg / L).

[0155] Comparative Example 2: Expression strain changed

[0156] The pET-32a plasmid containing the gene encoding the codon-optimized fusion protein TSF was transformed into Rosetta (DE3) competent cells or Origami B (DE3) competent cells. Positive clones were selected and induced to express the protein under the same conditions as in Example 1. Equal amounts of bacterial cells were collected, sonicated, and the supernatant and precipitate were separated. SDS-PAGE analysis was performed to compare the soluble proportion and total amount of the target protein in each strain.

[0157] Results: In both host cell lines, the target fusion protein TSF was solublely expressed. The expression level of TSF in Rosetta (DE3) cells reached 18 mg / mL, and in Origami B (DE3) cells it reached 10 mg / mL, but the total protein expression was significantly lower than in Example 1. Furthermore, the relatively higher yield of TSF from Rosetta (DE3) cells resulted in poor assembly when used for encapsulating glucosinolates. Figure 18 It is difficult to complete the encapsulation of glucosinolates.

[0158] Comparative Example 3: TSF protein expression was induced at 16℃.

[0159] The procedure was the same as in Example 1, except that the induction temperature was changed to 16°C and 37°C respectively. The bacterial cells were collected, the protein was purified, and the final protein yield was determined.

[0160] Conclusion: The yield of the fusion protein TSF induced at 16℃ was approximately 25 mg / L, and the yield of the fusion protein TSF induced at 37℃ was approximately 11 mg / L. Figure 19 The protein was well expressed in a soluble manner, but the expression level was low, far below the 50 mg / L in Example 1. Therefore, induction at 20°C was the preferred method. The produced fusion protein TSF can be used for encapsulation with glucosinolates.

[0161] Comparative Example 4: Different Embedding Steps

[0162] The rest is the same as in Example 1, except that glucosinolates and myrosinase are added together to the TSF protein prepared in Example 1; the step of separating and embedding glucosinolates and myrosinase is omitted. After mixing and stirring for 10 minutes, the protein concentration is adjusted to 30 mg / mL, and then 5 mg of Zn is added. 2+ Inducing the formation of hydrogels.

[0163] Results: Isothiocyanates were rapidly formed within 1 hour, and formation ceased thereafter. Figure 10 In Example 1, isothiocyanate has a continuous formation over 12 hours, which is more conducive to the utilization of unstable substances.

[0164] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A TSF fusion protein, characterized in that, From the N-terminus to the C-terminus, it includes: shrimp tropomyosin subunit, linker peptide, and shrimp ferritin subunit; The amino acid sequence of the shrimp tropomyosin subunit is shown in SEQ ID NO:1; The amino acid sequence of the shrimp ferritin subunit is shown in SEQ ID NO:3; The linker peptide is (GGGGS)n; n is an integer from 1 to 10.

2. The nucleic acid encoding the TSF fusion protein of claim 1.

3. The nucleic acid according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid encoding the TSF fusion protein is shown in SEQ ID NO:

6.

4. A hydrogel, characterized in that, The encapsulation product of the TSF fusion protein according to claim 1 after encapsulating glucosinolates and black myrosinase in Zn 2+ It is prepared by mixing under the condition that it exists.

5. The hydrogel according to claim 4, characterized in that, The concentration ratio of the TSF fusion protein to glucosinolate according to claim 1 is 1:(50~500).

6. The hydrogel according to claim 4, characterized in that, The Zn 2+ The concentration ranges from 2 mg / mL to 8 mg / mL.

7. The hydrogel according to claim 4, characterized in that, The conditions for encapsulating glucosinolates in the TSF fusion protein according to claim 1 are ultrasonic treatment for 30 to 120 minutes.

8. The hydrogel according to claim 4, characterized in that, The concentration of the black mycosesin is 8 mM to 12 mM; The concentration of the TSF fusion protein in the embedded product is 28 mg / mL to 32 mg / mL.

9. The hydrogel according to any one of claims 4 to 8, characterized in that, The host cell for the TSF fusion protein is BL21(DE3), the induction temperature is 20°C, and the nucleotide sequence of the nucleic acid encoding the TSF fusion protein is shown in SEQ ID NO:

6.

10. The use of the TSF fusion protein of claim 1 and / or the hydrogel of any one of claims 4 to 9 in the preparation of anticancer, antibacterial and / or antiviral drugs.

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