Light-responsive assembly proteins and their applications

Abstract

The present invention relates to a series of light-responsive assembly proteins and their applications. The present invention designs and expresses a series of proteins in Escherichia coli containing specific aminoacyl-tRNA synthetases and in the presence of AzoF, and then obtains proteins in the screening that can disaggregate the polymer or reduce affinity under 340nm light, and can restore its aggregation state or interaction mode under 420nm light. The protein obtained by screening in this application has a small molecular weight, good stability, and high expression level, and can be used as a label to convert monomeric target proteins into light-sensitive fusion polymeric proteins. At the same time, the light-responsive assembly protein of the present application can be applied to the fields of hydrogels and biosensors, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of protein technology, and in particular to a class of light-responsive assembly proteins and applications thereof. Background Art

[0002] In nature, light-responsive proteins play essential roles in many critical life processes by regulating membrane potential, protein interactions, and protein conformational changes. The use of light as a precise, reversible, and non-invasive control signal has enabled significant advances in optogenetics and photobiochemistry, particularly in controlling molecular and cellular processes such as protein localization and neuronal activity. Furthermore, light-responsive switches have been applied to the extracellular space for the creation of responsive biohybrid materials. The photosensitivity of these proteins is often mediated by pigments that absorb light at specific wavelengths, triggering conformational changes in the protein and initiating downstream signaling, such as the opening of ion channels or alterations in protein interactions. However, nature offers a limited selection of these proteins. Furthermore, due to the inherently complex photoreceptor mechanisms of these naturally derived proteins, precise control of their photoreceptor spectra and kinetics, as well as their effective use in exogenous systems, remains challenging.

[0003] Protein design has emerged as a powerful tool for creating proteins with novel structures and functions not yet explored in nature. Significant progress has been made in recent years, such as the design of novel binders and protein assemblies with diverse structures. While computational approaches have been attempted to engineer new photoresponsive proteins, these approaches are primarily based on existing natural photoresponsive proteins. Introducing new conformational switching functions into these proteins is complex and limited, and the photoresponse in these systems can be unpredictable and difficult to modulate. Furthermore, previous protein design strategies have primarily used the canonical 20 amino acids as primary building blocks, leaving the potential amino acid chemical space largely unexplored. While computational design methods have been adapted to incorporate noncanonical amino acids into proteins for structural optimization, the technology for accurately positioning these noncanonical amino acids at key functional sites remains in its infancy.

[0004] Genetic code expansion technology allows for the integration of non-canonical amino acids into proteins by reassigning codons through orthogonal aminoacyl-tRNA synthetase / tRNA pairing, thereby providing exceptional control over protein function. This approach exploits the unique properties of non-canonical amino acids to expand the chemical capabilities of proteins beyond the traditional twenty amino acids, thereby introducing novel functionalities such as light sensing and bioorthogonal chemistry into target proteins. Azobenzene, a classic photoswitchable group, exhibits reversible, light-induced, wavelength-selective cis / trans isomerization with high quantum yields. Under 340 nm light, phenylalanine-4'-azobenzene (AzoF), a photosensitive non-natural amino acid, can photoisomerize from the more stable E-isomer to the Z-isomer and revert to the E-isomer under 420 nm light. It has been widely used in biological settings to control protein motion by chemically linking adjacent residues or to genetically engineer it as a non-canonical amino acid to control DNA binding, fluorescent protein function, and enzyme activity. In previous studies, the incorporation site of AzoF was determined by a semirational approach that did not fully optimize the correlation between AzoF conformational adjustments and subsequent functional outcomes. Summary of the Invention

[0005] The technical purpose of the present application is to provide a class of protein polymers assembled in response to light using AzoF as a design target and related applications.

[0006] In one aspect, the present invention provides a light-responsive assembly protein containing phenylalanine-4'-azobenzene or a mutant thereof, which is in a disaggregated state under 340 nm wavelength light and in an aggregated state under 420 nm wavelength light, wherein the light-responsive assembly protein is selected from the following:

[0007] (i) a homomer selected from the group consisting of:

[0008] LRO-C2-5 dimer, a monomer of which comprises the amino acid sequence of SEQ ID No.: 1;

[0009] LRO-C2-35 dimer, a monomer of which comprises the amino acid sequence of SEQ ID No.: 2;

[0010] LRO-C3-7 trimer, the monomer of which comprises the amino acid sequence of SEQ ID No.: 3;

[0011] LRO-C3-37 trimer, a monomer of which comprises the amino acid sequence of SEQ ID No.: 4;

[0012] LRO-C3-64 trimer, a monomer of which comprises the amino acid sequence of SEQ ID No.: 5;

[0013] LRO-C4-13 tetramer, a monomer of which comprises the amino acid sequence of SEQ ID No.: 6;

[0014] An LRO-C5-1 pentamer, a monomer of which comprises the amino acid sequence of SEQ ID No.: 7, and (ii) a heterodimer comprising an A chain and a V chain selected from the group consisting of:

[0015] LRD-2 heterodimer: its A chain comprises the amino acid sequence of SEQ ID No.: 8, and its V chain comprises the amino acid sequence of SEQ ID No.: 9;

[0016] LRD-7 heterodimer: its A chain comprises the amino acid sequence of SEQ ID No.: 10, and its V chain comprises the amino acid sequence of SEQ ID No.: 11;

[0017] LRD-10 heterodimer: its A chain includes the amino acid sequence of SEQ ID No.: 12, and its V chain includes the amino acid sequence of SEQ ID No.: 13,

[0018] The mutants refer to sequences having at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity with the above sequences SEQ ID No.: 1-13, respectively, provided that the phenylalanine-4'-azobenzene contained in each sequence does not change.

[0019] In a specific embodiment,

[0020] The amino acid sequence of the LRO-C2-5 dimer monomer is SEQ ID No.: 1;

[0021] The amino acid sequence of the LRO-C2-35 dimer monomer is SEQ ID No.: 2;

[0022] The amino acid sequence of the LRO-C3-7 trimer monomer is SEQ ID No.: 3;

[0023] The amino acid sequence of the LRO-C3-37 trimer monomer is SEQ ID No.: 4;

[0024] The amino acid sequence of the LRO-C3-64 trimer monomer is SEQ ID No.: 5;

[0025] The amino acid sequence of the LRO-C4-13 tetramer monomer is SEQ ID No.: 6;

[0026] The amino acid sequence of the LRO-C5-1 pentamer monomer is SEQ ID No.: 7;

[0027] The amino acid sequence of the A chain of the LRD-2 heterodimer is SEQ ID No.: 8, and the amino acid sequence of the V chain is SEQ ID No.: 9;

[0028] The amino acid sequence of the A chain of the LRD-7 heterodimer is SEQ ID No.: 10, and the amino acid sequence of the V chain is SEQ ID No.: 11;

[0029] The amino acid sequence of the A chain of the LRD-10 heterodimer is SEQ ID No.: 12, and the amino acid sequence of the V chain is SEQ ID No.: 13.

[0030] In a specific embodiment, the N-terminus of each of the above sequences SEQ ID No.: 1-13 further includes the sequence MG, and / or the C-terminus of each of the above amino acid sequences further includes a tag sequence for purification, such as 6*His, MBP, GST, HA, c-Myc, Flag, etc., which can be connected to the C-terminus of each sequence through a linker, and the linker is selected from one or more of GS, GG, GGG, GGS or GSG, and the number of repetitions of each linker is 1-20 times, for example, the linker is (GGS) 1-5 .

[0031] In a specific embodiment, the amino acid sequence of the LRO-C2-5 dimer monomer is SEQ ID No.: 30;

[0032] The amino acid sequence of the LRO-C2-35 dimer monomer is SEQ ID No.: 31;

[0033] The amino acid sequence of the LRO-C3-7 trimer monomer is SEQ ID No.: 32;

[0034] The amino acid sequence of the LRO-C3-37 trimer monomer is SEQ ID No.: 33;

[0035] The amino acid sequence of the LRO-C3-64 trimer monomer is SEQ ID No.: 34;

[0036] The amino acid sequence of the LRO-C4-13 tetramer monomer is SEQ ID No.: 35;

[0037] The amino acid sequence of the LRO-C5-1 pentamer monomer is SEQ ID No.: 36;

[0038] The amino acid sequence of the A chain of the LRD-2 heterodimer is SEQ ID No.: 37, and the amino acid sequence of the V chain is SEQ ID No.: 40;

[0039] The amino acid sequence of the A chain of the LRD-7 heterodimer is SEQ ID No.: 38, and the amino acid sequence of the V chain is SEQ ID No.: 41;

[0040] The amino acid sequence of the A chain of the LRD-10 heterodimer is SEQ ID No.: 39, and the amino acid sequence of the V chain is SEQ ID No.: 42.

[0041] On the other hand, the present invention provides use of the above homomer in preparing a photoresponsive hydrogel.

[0042] In a specific embodiment, the photoresponsive hydrogel can switch between the gel and solution states in response to light, for example, it presents a gel state under light with a wavelength of 420 nm and a solution state under light with a wavelength of 340 nm.

[0043] In a specific embodiment, the homomer is the above-mentioned LRO-C5-1 pentamer.

[0044] In another aspect, the present invention provides a method for constructing a photoresponsive hydrogel system, the method comprising:

[0045] 1) constructing a plasmid for expressing the fusion protein SpyTag-MBP-LRO-C5-1, and expressing and purifying it to obtain the fusion protein SpyTag-MBP-LRO-C5-1, wherein the fusion protein SpyTag-MBP-LRO-C5-1 comprises, from N-terminus to C-terminus, a Spy-tag sequence, linker 1, an MBP sequence, linker 2, and the LRO-C5-1 monomer sequence described above; and

[0046] A plasmid for expressing a homodimeric Dimer-SpyCatcher protein is constructed, and expression and purification are performed to obtain a homodimeric Dimer-SpyCatcher protein. The monomer sequence of the homodimeric Dimer-SpyCatcher protein comprises, from the N-terminus to the C-terminus, a homodimer sequence, a linker3, and a Spy-cartcher sequence.

[0047] 2) The fusion protein SpyTag-MBP-LRO-C5-1 obtained in step 1) is mixed with the homodimer Dimer-SpyCatcher protein at a molar ratio of 1:2-3, so that the protein ratio in the final solution is between 6% and 20%, and a gel is formed after reaction.

[0048] In a specific embodiment, the linker1, linker2, and linker3 are each independently selected from a repeating sequence of one or more of GS, GG, GGG, GGS, or GSG, and the number of repetitions may be 1-20 times.

[0049] In a specific embodiment, the amino acid sequence of SpyTag-MBP-LRO-C5-1 is SEQ ID No.: 14, and the amino acid sequence of the homodimer Dimer-SpyCatcher protein monomer is SEQ ID No.: 15.

[0050] In a specific embodiment, in step 2), the molar ratio of SpyTag-MBP-LRO-C5-1 to Dimer-SpyCatcher is 1:2.5, and the reaction time is 1 hour.

[0051] In another aspect, the present invention provides a photoresponsive hydrogel system comprising the following two proteins in an aqueous buffer dispersion medium:

[0052] The fusion protein SpyTag-MBP_LRO-C5-1 comprises the SpyTag sequence, linker1, MBP sequence, linker2, and LRO-C5-1 monomer sequence from N-terminus to C-terminus; and

[0053] Homodimeric Dimer-SpyCatcher protein: Its monomer sequence contains homodimer sequence, linker3, and SpyCatcher sequence from N-terminus to C-terminus.

[0054] In a specific embodiment, the buffer only provides an aqueous environment for the formation of the gel, and thus its selection is not limited, and can be, for example, conventional PBS or TBS buffer.

[0055] In a specific embodiment, the linker1, linker2, and linker3 are each independently selected from a repeating sequence of one or more of GS, GG, GGG, GGS, or GSG, and the number of repetitions may be 1 to 20 times.

[0056] In a specific embodiment, the amino acid sequence of the fusion protein SpyTag-MBP_LRO-C5-1 is SEQ ID No.: 14, and the amino acid sequence of the homodimer Dimer-SpyCatcher protein monomer is SEQ ID No.: 15.

[0057] In a specific embodiment, the photoresponsive hydrogel system converts from a gel state to a liquid state under 340 nm light irradiation and returns to a gel state under 420 nm light irradiation.

[0058] In a specific embodiment, for the photoresponsive hydrogel system, when the protein-solvent ratio is 10% w / v, the modulus in the gel state is about 10 times that in the liquid state.

[0059] In another aspect, the present invention provides use of the above-mentioned photoresponsive hydrogel system in preparing cell culture medium.

[0060] In another aspect, the present invention provides a use of the heterodimer in preparing a light-responsive extracellular molecule sensor.

[0061] In a specific embodiment, the heterodimer is LRD-7.

[0062] In a specific embodiment, the A chain of LRD-7 is homodimerized to form a D-7A dimer protein, which serves as a light-responsive extracellular molecule sensor. The V chain of the LRD-7 heterodimer is further fused to the N-terminus of the erythropoietin receptor EpoR to serve as a receptor on the cell membrane of the light-responsive extracellular molecule sensor.

[0063] In a specific embodiment, the amino acid sequence of the extracellular domain of EpoR constructed by fusing the V chain of the LRD-7 heterodimer to the N-terminus of EpoR is SEQ ID No.: 23, and the amino acid sequence of the A chain dimer monomer of the homodimer D-7A is SEQ ID No.: 22.

[0064] In a specific embodiment, the light-responsive extracellular molecular sensor can activate cell signaling pathways (e.g., JAK / STAT, PLCG, MAPK) in response to light, thereby promoting cell secretion of reporter gene products (e.g., SEAP enzymes). Specifically, by treating the molecular sensor with 340 nm light, the cell signaling pathway (e.g., JAK / STAT, PLCG, MAPK) can be controlled to inhibit cell secretion of reporter gene products (e.g., SEAP enzymes), while by treating the molecular sensor with 420 nm light, the cell signaling pathway (JAK / STAT, PLCG, MAPK) can be activated to promote cell secretion of reporter gene products (e.g., SEAP enzymes).

[0065] Beneficial effects

[0066] The present invention precisely designed a series of C-symmetric homologous 2-, 3-, 4-, and 5-mers (respectively LRO-C2-5, LRO-C2-35, LRO-C3-7, LRO-C3-37, LRO-C3-64, LRO-C4-13, and LRO-C5-1) and heterologous 2-mer proteins (respectively LRD-2, LRD-7, and LRD-10), and expressed this series of proteins in Escherichia coli containing specific aminoacyl-tRNA synthetases and in the presence of AzoF. Subsequently, proteins that can disaggregate or reduce the affinity of the polymer under 340nm light and restore its aggregation state or interaction mode under 420nm light were obtained in the screening. Among them, the affinity difference of LRD-7 is close to 177 times. In addition, the proteins obtained by screening have a small molecular weight, good stability, and high expression level, and can be used as tags to convert monomeric target proteins into light-sensitive fusion polymeric proteins.

[0067] Based on the light-controlled pentamers in this series of light-sensitive proteins, this application further designed and prepared a light-sensitive protein hydrogel. Under irradiation with light of a wavelength of 340nm, this hydrogel transforms from a gel (gel) to a solution (sol) state. Under irradiation with light of 420nm, it transforms from a solution (sol) to a gel (gel). Rotational rheometer testing showed that the storage modulus changed by nearly 10 times, and this transformation can be repeated without a significant decrease in properties.

[0068] Furthermore, in this application, a protein that controls the conduction of cell signaling pathways is designed by utilizing heterodimer fusion. This protein can transmit to the three intracellular signaling pathways of JAK / STAT, PLCG, and MAPK through the modified EpoR on the cell surface, and is specifically manifested in the secretion and expression of SEAP enzyme. The relevant pathways can be controlled by 340nm light to inhibit the secretion of related enzymes, and can be restored and activated after irradiation with 420nm light. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Schematic diagram showing the design, synthesis, photoresponsive chromatography principle and application of the light-responsive assembly protein in this application in light-responsive gels and extracellular molecular sensors.

[0070] Figure 2 The photoresponsive gel chromatography results of LRO-C2-5, LRO-C3-7, LRO-C4-13, and LRO-C5-1 are shown.

[0071] Figure 3 Model structure diagrams showing the LRO-C2-5, LRO-C3-7, LRO-C4-13, LRO-C5-1, LRD-2, LRD-7 and LRD-10 designs.

[0072] Figure 4 Graphs showing the analytical ultracentrifugation results for LRO-C2-5, LRO-C3-7, LRO-C4-13, and LRO-C5-1.

[0073] Figure 5 The crystal structures of LRO-C2-35 at different pH conditions, as well as the model structures of LRO-C3-37 and LRO-C3-64, and the photoresponsive gel chromatography results of the three proteins are shown. Among them, a. Crystal structure of LRO-C2-35 at pH 8.0; b. Crystal structure of LRO-C2-35 at pH 4.5; c. Photoresponsive gel chromatography results of LRO-C2-35; d. Model structure of LRO-C3-37; e. Model structure of LRO-C3-64; f. Photoresponsive gel chromatography results of LRO-C3-37; g. Photoresponsive gel chromatography results of LRO-C3-64.

[0074] Figure 6 The SDS-PAGE gel images of some proteins involved in the present invention are shown. A shows the SDS-PAGE gel image of homomers; B shows the SDS-PAGE gel image of heterodimers.

[0075] Figure 7 A schematic diagram showing the construction model and light-responsiveness process for preparing a photoresponsive hydrogel. A shows the construction of each protein molecule in the hydrogel; B shows the changes in the hydrogel state under different light conditions.

[0076] Figure 8 Shown is the morphology switching of the photoresponsive hydrogel upon 340 / 420 nm light irradiation.

[0077] Figure 9 Figure 3. Storage modulus of a photoresponsive hydrogel in two states and during switching between these two states. A: Storage modulus in the gel state at a protein-solvent ratio of 10% w / v and in the liquid state after 340 nm irradiation. B: Results of the gel irradiated with alternating 340 / 420 nm irradiation.

[0078] Figure 10 The results of photoresponsive gel chromatography of LRD-2 / 7 / 10 with and without GFP tag are shown.

[0079] Figure 11 A graph showing the results of a concentration gradient BLI experiment on the interaction between LRD-2 / 7 / 10-A and LRD-2 / 7 / 10-V.

[0080] Figure 12 Graphs showing the light responses of LRD-2 / 7 / 10-A and LRD-2 / 7 / 10-V and the BLI results of AzoF A or F mutations.

[0081] Figure 13 Schematic diagram showing the mechanism of action of light-responsive extracellular molecule sensors.

[0082] Figure 14 The graph shows the changes in the ability of D7A to activate SEAP secretion when the protein is treated at 340 nm and the cells are treated at 420 nm.

[0083] Figure 15 Shown are the crystal structure of the photoresponsive assembly protein and the verification of the AzoF electron cloud density. DETAILED DESCRIPTION

[0084] The technical solutions of the present application are described in detail below through specific implementation methods so that those skilled in the art can fully understand the present invention. However, these embodiments are not intended to limit the scope of the present application.

[0085] the term

[0086] In this application, "assembled protein" and "protein aggregate" are used interchangeably to refer to an aggregate formed by the assembly of identical (homomer) or different protein monomers (heteromer).

[0087] As used herein, "SEAP" means secreted embryonic alkaline phosphatase.

[0088] The Spy-tag sequence, MBP sequence, homodimer sequence, and Dimer-SpyCatcher (containing 5 GGS) used in the preparation of the hydrogel in this article can be found in the document PMID: 38289949.

[0089] Example 1:

[0090] Protein design, construction, preparation and purification:

[0091] The designed homologous multimers (LROs), including monomer coding sequences for C2 (45), C3 (55), C4 (43), and C5 (45) symmetric C3 polymers, were synthesized (Azenta) between the NcoI and XhoI residues of pET-28a. The codon for AzoF was TAG. The synthesized plasmids were then chemically transformed into bacterial strains containing the AzoF aminoacyl-tRNA synthetase and specific tRNA through incubation, heat shock, recovery, and plating. The strain was transformed into a conventional BL21 (DE3) (New England Biolabs) by transforming a plasmid containing a specific AzoF aminoacyl-tRNA synthetase and tRNA AzoRS4 (ref: / / doi.org / 10.1002 / adfm.202011276) into the competent medium, and then the competent medium was prepared by the Inoue method to obtain the AzoF expression competent BL21 (DE3, AzoRS4). After growth on the plates, monoclonal bacteria were picked and cultured in liquid LB at 37°C, 220 rpm. After the OD600 reached 0.8, 0.3 mM IPTG (Sangon Biotech) and a final concentration of 0.1 mg / ml of the unnatural amino acid AzoF (WuXi AppTec) were added. The shaker temperature was lowered to 22°C and the speed was unchanged to express the target protein. 15 hr after induction, the cells were collected by centrifugation at 10,000g for 5 min and resuspended in approximately 15 ml of TBS (150 mM NaCl, 20 mM Tris pH 7.4) buffer. The cells were then ultrasonically disrupted at 80% power for 15 min and centrifuged at 17,000g to remove the precipitate. The supernatant was purified using Ni-NTA (QIAGEN) affinity purification and gel chromatography (Union-Biotech) to obtain the target protein, and the presence of the target protein was confirmed by SDS-PAGE (Genscript).

[0092] The above methods are also applicable to the construction of each plasmid, expression and purification of target proteins in Examples 2 and 3 below.

[0093] Analysis of the photoresponsive properties of protein aggregates

[0094] The prepared protein was divided into two 2 ml portions, each containing one portion. One portion was directly analyzed for peak position by gel chromatography, while the other portion was placed in a 1.5 ml centrifuge tube and irradiated with a 340 nm laser at a diameter of 1 cm and analyzed for its deaggregation properties by gel chromatography. The analytical column was a Superdex 75 increase, 10 / 300 GL, and the chromatograph was a Yonglian Bio UEV25M. The sample volume was 1.8 ml, the flow rate was 0.8 ml / min, and the temperature was 24°C. The protein loading was approximately 0.3 mg. After comprehensively determining the peak position and light responsiveness, further testing was performed to determine the protein's ability to recover aggregation after irradiation at 420 nm.

[0095] like Figure 2 As shown, the screening experiment yielded four proteins (LRO-C2-5, LRO-C3-7, LRO-C4-13, and LRO-C5-1) that exhibit distinct peak positions in response to 340 nm and 420 nm irradiation. Their designed monomer sequences are SEQ ID No.: 1, SEQ ID No.: 3, SEQ ID No.: 6, and SEQ ID No.: 7, respectively. These four proteins moved closer to the monomer peak position (15-16 ml) after 340 nm irradiation. Samples treated with 340 nm irradiation with a 420 nm laser were able to restore their natural peak positions.

[0096] Furthermore, an analytical ultracentrifuge (Beckman, Optima AUC03061703) was used at a protein concentration of approximately 0.4 mg / ml, 42,000 rpm, and a collection interval of 20 seconds. Using a resin centerpiece, 390 μl of sample was added to the experimental group, and 400 μl of TBS was added to the blank control group. The sample cell was sealed according to the operating procedures and centrifuged for 16 hours. After the experiment, the continuous c(M) distribution model was used to analyze the changes in the sedimentation coefficient of the screened proteins. Figure 4 As shown, the four proteins can switch between the aggregated state and the monomeric state, respectively, demonstrating their light-responsive properties.

[0097] Through experimental verification, the present invention has obtained one homologous 2, 3, 4, and 5 aggregates that can respond well to light, namely LRO-C2-5, LRO-C3-7, LRO-C4-13, and LRO-C5-1. Their model diagrams were plotted using ChimeraX, and the results are as follows Figure 3 As shown in (first row), SDS-PAGE analysis results are as follows Figure 6 As shown in A.

[0098] In addition, three LRO proteins were designed and synthesized to respond to light, but the effect was slightly worse. Figure 5As shown, the aggregation state of LRO-C2-35 (its monomer sequence is shown in SEQ ID No.: 2) is greatly affected by concentration and is less affected by 340nm light treatment. LRO-C3-37 (its monomer sequence is shown in SEQ ID No.: 4) tends to aggregate after 420nm light treatment, and LRO-C3-64 (its monomer sequence is shown in SEQ ID No.: 5) has a certain degree of aggregation after both light treatments.

[0099] The amino acid sequences of the homomeric monomers prepared above with MG at the N-terminus and histidine tags at the C-terminus are as follows:

[0100] LRO-C2-5 dimer monomer: SEQ ID No.: 30;

[0101] LRO-C2-35 dimer monomer: SEQ ID No.: 31;

[0102] LRO-C3-7 trimer monomer: SEQ ID No.: 32;

[0103] LRO-C3-37 trimer monomer: SEQ ID No.: 33;

[0104] LRO-C3-64 trimer monomer: SEQ ID No.: 34;

[0105] LRO-C4-13 tetramer monomer: SEQ ID No.: 35;

[0106] LRO-C5-1 pentamer monomer: SEQ ID No.: 36.

[0107] Example 2: Preparation and property detection of photoresponsive hydrogel

[0108] The photoresponsive pentamer LRO-C5-1 obtained in Example 1 was used to prepare the photoresponsive hydrogel.

[0109] like Figure 7As shown, the maltose binding protein (MBP) protein was first added to the N-terminus of the LRO-C5-1 monomer sequence (its monomer sequence is shown in SEQ ID No.: 7). On the one hand, the addition of MBP increased protein expression, and on the other hand, increased the size of the protein, improving the modulus of the gel. A SpyTag tag was fused to the N-terminus of MBP for covalent attachment to prepare a hydrogel. The complete pentameric fragment was named SpyTag-MBP-LRO-C5-1 (ST-MBP_LRO-C5) (SEQ ID No.: 14). The amino acid sequence was codon-optimized for expression in Escherichia coli. The optimized encoding nucleic acid sequence was constructed between NcoI and XhoI of pET28a. The constructed plasmid was transformed into the AzoF expression competent BL21 (DE3, AzoRS4) for expression and purification.

[0110] The homodimeric protein Dimer-SpyCatcher protein required for hydrogel preparation is a dimer protein reported in the literature (C2-(GGS)5-SpyCatcher (C2-SC), also known as homodimeric Dimer-SpyCatcher protein, SEQ ID No.: 15).

[0111] After plasmid construction and protein purification, ST-MBP_LRO-C5 and C2-SC proteins were obtained and concentrated to 200 mg / ml in TBS (150 mM NaCl, 20 mM Tris pH 7.4) using 10 kD ultrafiltration tubes (Millipore). During ultrafiltration, the solution medium was replaced with TBS. ST-MBP_LRO-C5 and C2-SC proteins were then mixed at a molar ratio of 1:2.5, resulting in a final solution with a combined ST-MBP_LRO-C5 and C2-SC protein fraction of 6%-20% w / v. After approximately 1 hour of reaction between the SpyTag and SpyCatcher, a gel was formed.

[0112] like Figure 8 As shown, the prepared hydrogel has the characteristics of changing its internal molecular structure and undergoing gel-liquid transformation under two wavelengths of light. It transforms from gel to liquid under 340nm light and returns to gel under subsequent 420nm light.

[0113] Determination of the storage modulus of photoresponsive hydrogels

[0114] The storage modulus of the hydrogel in two states was tested using a rotational rheometer (TA-Waters, ARES-G2), and the gap value of a single test was 0.8 mm.

[0115] like Figure 9 As shown in A, when the protein-solvent ratio is 10% w / v, the modulus in the gel state is about 75-110 Pa. After irradiation at 340 nm, the modulus in the liquid state is 7-10 Pa, and the modulus changes by nearly 10 times. In addition, experiments were also conducted in which the gel was irradiated alternately at 340 / 420 nm. Figure 9 As shown in B, the storage modulus changes uniformly and stably under repeated switching between gel and liquid states.

[0116] Example 3: Photoresponsive gel chromatography, interaction BLI assay, concentration gradient assay, and mutation BLI assay of LRD-2 / 7 / 10 heterodimers

[0117] In this example, the screening and effect verification of light-responsive heterodimers were carried out.

[0118] The two halves of the designed heterodimer were co-expressed in the same vector. The expressed A chain proteins (LRD-2A, 7A, and 10A) contained an AzoF-containing portion with a 6*His tag at the tail for affinity purification. The V chain proteins (LRD-2V, 7V, and 10V) lacked AzoF. A monomeric GFP protein (mBaojin; PMID: 38409224) with the amino acid sequence of SEQ ID No. 28 was added to the C-terminus of the V chain via a linker (GGS). The addition of GFP facilitated visualization and increased the molecular weight difference between the A and V chains, facilitating the shift in peak position observed under 340 nm illumination using gel chromatography.

[0119] Azenta constructed 35 constructs and screened them by gel chromatography, obtaining three light-responsive proteins, LRD-2 / 7 / 10 (the A chain amino acid sequences of LRD-2 / 7 / 10 are SEQ ID No.: 8, SEQ ID No.: 10, and SEQ ID No.: 12, and the corresponding sequences with MG and 6*His tags are SEQ ID No.: 37-39, respectively; the V chain amino acid sequences of LRD-2 / 7 / 10 are SEQ ID No.: 9, SEQ ID No.: 11, and SEQ ID No.: 13, and the corresponding sequences with MG and 6*His tags are SEQ ID No.: 40-42, respectively). The light-responsive gel chromatography analysis results of the three groups of proteins with and without GFP are shown in the figure below. Figure 10 shown.

[0120] The A and V chains of these three proteins were expressed separately, and an AVI tag (GLNDIFEAQKIEWHE SEQ ID No.: 29) was added to the C-terminus of the V chain constructs (LRD-2V, 7V, and 10V-AVI) for BLI testing. BLI experiments were performed using biotinylated LRO-2V-AVI, 7V-AVI, and 10V-AVI, as well as LRO-2A, 7A, and 10A, and AzoF site mutant proteins (the AzoF amino acid site was mutated to alanine or phenylalanine). After gel chromatography purification, the three AVI-tagged proteins were replaced by ultrafiltration into 80mM NaCl, 20mM Tris pH 7.4, and then biotinylated using the BirA-RT Kit (Avidity). The labeled sample concentration was 100μM, and the reaction was incubated at 25°C for 2 hours. 5μg of BirA biotinylase was added to each reaction. Subsequently, the protein peaks were desalted and purified by gel chromatography, and the protein peaks were collected and concentrated to approximately 1mg / ml using ultrafiltration tubes. After labeling, BLI experiments were performed using an SA probe (ForteBio) on an OCTET RED96E (ForteBio) instrument. Ligand binding to the probe was at a concentration of 10 μg / ml. LRO-2A, 7A, and 10A were diluted two-fold using a maximum concentration of 900 nM. Proteins were diluted in TBST (150 mM NaCl, 20 mM Tris pH 7.4, 0.05% Tween-20), and the working volume per well for BLI was 200 μl.

[0121] like Figure 11 As shown in the figure, orthogonal experiments showed that LRD-2A / 7A / 10A interacted with the corresponding LRD-2V-AVI / 7V-AVI / 10V-AVI with good specificity. Even at a concentration of 900 nM, the response value of the interaction with non-corresponding proteins was very weak. The concentration gradient results of LRD-2A / 7A / 10A showed that the K d The value is around 20nM; the LRD-2A / 7A / 10A protein concentration used in the light response experiment is 225nM.

[0122] like Figure 12 As shown in Figure 2, the 340 nm photoresponse K of LRD-7 d The maximum reduction factor can reach 177 times, and the K of LRD-2 / 10 d The reduction factor was relatively low; the protein concentration used in the mutation experiment was 900 nM. The results showed that mutating the AzoF amino acid site in LRD-2A / 7A to alanine or phenylalanine using a point mutagenesis kit (TakaRa) could eliminate its interaction with the V protein, while the two mutations in LRD-10A significantly weakened the interaction.

[0123] In addition, the model of the heterodimer constructed in this example is shown in Figure 3 (second row), the SDS-PAGE analysis results of each protein monomer are shown in Figure 6 In B.

[0124] Example 4: Preparation of a light-responsive extracellular molecule sensor

[0125] In this example, the light-responsive heterodimer LRD-7 in Example 3 was selected to further prepare a light-responsive extracellular molecular sensor. The experimental principle is shown in Figure 13 .

[0126] 1. The LRD-7V protein in the heterodimer was constructed to the N-terminus of EpoR to obtain the extracellular segment protein 7V-Epo (SEQ ID No.: 23). Specifically, the plasmid pSL889 (P sv40 -7V-EpoR-IL6BR int -pA, SEQ ID No.: 16 JAK / STAT pathway), pSL893 (P sv40 -7V-EpoR-VEGFR2 int -pA, SEQ ID No.: 17PLCG pathway) and pSLM112 (P sv40 -7V-EpoR-FGFR int -pA, SEQ ID No.: 18 MAPK pathway), by inserting the coding sequence of 7V between the BamHI and EcoRI restriction sites of the above vector; the amino acid sequences of the receptor proteins expressed by the above plasmids are SEQ ID No.: 19, SEQ ID No.: 20 and SEQ ID No.: 21, respectively.

[0127] 2. The LRD-7A protein was fused to a homodimer tag to form a fused Homodimer-7A (D7A) protein (SEQ ID No.: 22). Specifically, the D7A codon was optimized in E. coli and the encoding nucleic acid sequence was inserted between the NcoI and XhoI regions of the pET28a vector. The plasmid was then transformed into BL21 (DE3, AzoRS4) cells for protein expression and purification.

[0128] 3. The cells used were 293T cells, cultured in a humidified incubator containing 10% fetal bovine serum (WISENT) and 1% streptomycin / penicillin (Thermo Fisher) at 37°C and 5% CO2. The experiment was performed in a 24-well plate, with 50,000 HEK293T cells per well. After 12 hours of cell culture, transfection was performed. The JAK / STAT pathway was transfected using 200 ng of the receptor plasmid pSL889 and 100 ng of the STAT3 reporter gene plasmid pYW123 (O Stat3 -P hCMVmin -SEAP-pA SEQ ID No.: 24); PLCG pathway used 200ng receptor plasmid pSL893, 100ng NFAT reporter gene plasmid pSYQ034 ( NFAT -P min -SEAP-pA.SEQ ID No.: 25); MAPK pathway using 200ng receptor plasmid pSLM112, 100ng TetR-dependent reporter gene plasmid pMF111 (O Tet -P hCMVmin -SEAP-pA SEQ ID No.: 26) and 10 ng of TetR-Elk1 fusion protein expression plasmid pTetR-Elk1 (P hCMV -TetR-ELK1-pA SEQ ID No.: 27). Transfection was performed using a DNA:PEI (PolyScience) ratio of 3:1 (w / w). The medium was changed 12 hours after transfection.

[0129] 4. After the transfection medium was changed, the D7A protein from step 2 or the D7A protein treated with 340 nm was added to the supernatant of the transfected cells. Subsequently, the corresponding group of cells treated with 340 nm D7A protein were irradiated with 420 nm light 3 or 10 hr later (3 / 10h-420nm), irradiating each well for 30 s. Finally, 15 hr after the protein addition, the SEAP enzyme activity in the supernatant was measured using a SEAP enzyme activity assay kit (Abcam, AB133077).

[0130] The specific plasmid construction of the receptor protein and intracellular SEAP secretion expression pathway used in the experiment can be found in the literature (PMID: 29686358).

[0131] like Figure 14As shown, JAK / STAT3, PLCG, and MAPK pathway proteins were added at final concentrations of 3, 25, and 6 nM, respectively. The results showed that compared to the blank control (blank), SEAP enzyme activity was increased by 4.7, 4.8, and 20.4 times in the untreated D7A protein (Native) group. D7A protein-induced SEAP secretion decreased by 2.8, 2.7, and 3.3 times, respectively, when treated with 340 nm light. Treatment at the two time points restored SEAP secretion activity to varying degrees.

[0132] Example 5: Crystal Screening

[0133] In this example, some proteins involved in light-responsive assembly (including LRO-C2-5, LRO-C3-7, LRO-C4-13, LRO-C5-1, LRD-2 / 7) were screened using crystal screening kits including PEGRx (Hampton), Crystal Screen (Hampton), and Wizard Classic 1-4 (Rigaku). Protein crystals were picked using XRD (Rigaku) using loops of corresponding sizes, and the crystals were diffracted. After data collection, the designed model was used to analyze the structure using Phenix software based on molecular replacement. The results are shown in Figure 2. Figure 15 As shown in Figure 2, the obtained crystal structures are very close to the designed models, and AzoF has significant electron cloud density in all six resolved structures, among which the crystal structure of LRD-7 reaches AzoF presents a very clear double benzene ring molecular structure.

[0134] Note: The 340nm and 420nm light sources used above are about 1cm in diameter, and the 340nm is 240mW / cm 2 , 420nm is 1500mW / cm 2 The light source was held 2 cm from the sample being treated. Treatment time varied depending on the sample volume. For a 1ml protein solution, treatment time at 340 nm was 120 s, and at 420 nm was 60 s. Treatment time for hydrogels was similar to that for protein solutions, increasing the irradiation time based on sample volume.

[0135] The monomer sequences of each protein polymer (including 7 homomers and 3 heterodimers) involved in this application are as follows:

[0136]

[0137]

[0138] In the above sequence, X represents AzoF, the bold part (MG) represents the starting amino acid M and the connecting amino acid G, and the underlined part (GGSHHHHHH) represents the linker+6*His.

Claims

1. A light-responsive assembly protein containing phenylalanine-4'-azobenzene, which is in a disaggregated state under 340 nm wavelength light and in an aggregated state under 420 nm wavelength light, wherein the light-responsive assembly protein is selected from: LRO-C2-5 dimer, the amino acid sequence of its monomer is SEQ ID No.: 1; LRO-C2-35 dimer, the amino acid sequence of its monomer is SEQ ID No.: 2; LRO-C3-7 trimer, the amino acid sequence of its monomer is SEQ ID No.: 3; LRO-C3-37 trimer, the amino acid sequence of its monomer is SEQ ID No.: 4; LRO-C3-64 trimer, the amino acid sequence of its monomer is SEQ ID No.: 5; LRO-C4-13 tetramer, the amino acid sequence of its monomer is SEQ ID No.: 6; LRO-C5-1 pentamer, the amino acid sequence of its monomer is SEQ ID No.: 7; LRD-2 heterodimer, the amino acid sequence of its A chain is SEQ ID No.: 8, and the amino acid sequence of its V chain is SEQ ID No.: 9; LRD-7 heterodimer, the amino acid sequence of its A chain is SEQ ID No.: 10, and the amino acid sequence of its V chain is SEQ ID No.: 11; The LRD-10 heterodimer has an A chain amino acid sequence of SEQ ID No.: 12 and a V chain amino acid sequence of SEQ ID No.:

13.

2. A light-responsive assembly protein containing phenylalanine-4'-azobenzene, which is in a disaggregated state under 340 nm wavelength light and in an aggregated state under 420 nm wavelength light, wherein the light-responsive assembly protein is selected from: LRO-C2-5 dimer, the amino acid sequence of its monomer is SEQ ID No.: 30; LRO-C2-35 dimer, the amino acid sequence of its monomer is SEQ ID No.: 31; LRO-C3-7 trimer, the amino acid sequence of its monomer is SEQ ID No.: 32; LRO-C3-37 trimer, the amino acid sequence of its monomer is SEQ ID No.: 33; LRO-C3-64 trimer, the amino acid sequence of its monomer is SEQ ID No.: 34; LRO-C4-13 tetramer, the amino acid sequence of its monomer is SEQ ID No.: 35; LRO-C5-1 pentamer, the amino acid sequence of its monomer is SEQ ID No.: 36; LRD-2 heterodimer, the amino acid sequence of its A chain is SEQ ID No.: 37, and the amino acid sequence of its V chain is SEQ ID No.: 40; LRD-7 heterodimer, the amino acid sequence of its A chain is SEQ ID No.: 38, and the amino acid sequence of its V chain is SEQ ID No.: 41; The LRD-10 heterodimer has an A chain amino acid sequence of SEQ ID No.: 39 and a V chain amino acid sequence of SEQ ID No.:

42.

3. A method for constructing a photoresponsive hydrogel system, the method comprising: 1) constructing a plasmid for expressing the fusion protein SpyTag-MBP-LRO-C5-1, and expressing and purifying it to obtain the fusion protein SpyTag-MBP-LRO-C5-1, wherein the fusion protein SpyTag-MBP-LRO-C5-1 comprises, from N-terminus to C-terminus: a Spy-tag sequence, linker 1, an MBP sequence, linker 2, and a monomer sequence of the LRO-C5-1 pentamer as claimed in claim 1 or 2; and A plasmid for expressing a homodimeric Dimer-SpyCatcher protein is constructed, and expression and purification are performed to obtain a homodimeric Dimer-SpyCatcher protein. The monomer sequence of the homodimeric Dimer-SpyCatcher protein comprises, from the N-terminus to the C-terminus, a homodimer sequence, a linker3, and a Spy-cartcher sequence. 2) The fusion protein SpyTag-MBP-LRO-C5-1 obtained in step 1) is mixed with the homodimer Dimer-SpyCatcher protein at a molar ratio of 1:2-3, so that the protein ratio in the final solution is between 6% and 20%, and a gel is formed after reaction.

4. The method according to claim 3, wherein: The linker1, linker2, and linker3 are each independently selected from one or more of GS, GG, GGG, GGS, or GSG, and are repeated 1-20 times.

5. The method according to claim 3, wherein: The amino acid sequence of SpyTag-MBP-LRO-C5-1 is SEQ ID No.: 14, and the amino acid sequence of the homodimer Dimer-SpyCatcher protein monomer is SEQ ID No.:

15.

6. The method according to claim 3, wherein: In step 2), the molar ratio of SpyTag-MBP-LRO-C5-1 to Dimer-SpyCatcher was 1:2.5, and the reaction time was 1 hour.

7. A photoresponsive hydrogel system comprising the following two proteins in an aqueous buffer dispersion medium: The fusion protein SpyTag-MBP-LRO-C5-1 comprises the Spy-tag sequence, linker 1, MBP sequence, linker 2, and LRO-C5-1 monomer sequence from N-terminus to C-terminus; and Homodimeric Dimer-SpyCatcher protein: Its monomer sequence contains homodimer sequence, linker3, and Spy-cartcher sequence from N-terminus to C-terminus.

8. The photoresponsive hydrogel system according to claim 7, wherein: The amino acid sequence of the fusion protein SpyTag-MBP-LRO-C5-1 is SEQ ID No.: 14, and the amino acid sequence of the homologous dimer Dimer-SpyCatcher protein monomer is SEQ ID No.:

15.

9. Use of the photoresponsive hydrogel system according to claim 7 or 8 in preparing cell culture medium.

10. Use of the LRD-7 heterodimer as claimed in claim 1 or 2 in preparing a light-responsive extracellular molecule sensor. in, The light-responsive extracellular molecule sensor was prepared by fusing the V chain of the LRD-7 heterodimer to the N-terminus of EpoR and constructing an A chain dimer of the LRD-7 heterodimer as a tag.

11. The use according to claim 10, wherein: The amino acid sequence of the extracellular domain of EpoR constructed by fusing the V chain of the LRD-7 heterodimer to the N-terminus of EpoR is SEQ ID No.: 23, and the amino acid sequence of the A chain dimer monomer of the LRD-7 heterodimer is SEQ ID No.: 22.

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