Photoresponse assembly protein and application thereof

Abstract

The invention relates to a series of photoresponse assembly proteins and application thereof. According to the invention, a series of proteins are designed and expressed in escherichia coli containing specific aminoacyl-tRNA synthetase and in the presence of AzoF, and then proteins which can be subjected to polymer depolymerization or affinity reduction under 340nm illumination and can recover the aggregation state or interaction mode under 420nm illumination are obtained in screening. The protein obtained by screening is small in molecular weight, good in stability and high in expression quantity, and can be used as a tag to convert a monomer target protein into a light-sensitive fusion multimerization protein. Meanwhile, the photoresponse assembly protein can be applied to the fields of hydrogel and biosensors, and has a wide application prospect.

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 important roles in many key 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 led to significant advances in optogenetics and photobiochemistry, especially in the control of molecular and cellular processes such as protein localization and neuronal activity. In addition, light-responsive switches have been applied to the extracellular space for the creation of responsive biohybrid materials. The photoreceptive ability of these proteins is usually mediated by pigments that absorb light at specific wavelengths, triggering protein conformational changes that initiate downstream signaling, such as the opening of ion channels or changes in protein interactions. However, nature offers a limited selection of these proteins. In addition, due to the complex photoreceptive mechanisms inherent in these naturally derived proteins, it remains challenging to precisely control their photoreceptive spectra and kinetics, as well as to effectively use them in exogenous systems.

[0003] Protein design has evolved into a powerful tool to create proteins with novel structures and functions that have not been explored in nature. In recent years, significant progress has been made in this field, such as designing new binders and protein assemblies with diverse structures. Although attempts have been made to use computational methods to engineer new light-responsive proteins, these approaches are mainly based on existing natural light-responsive proteins. Introducing new conformational switching functions in these proteins is complex and limited, and the photoresponse in these systems can be unpredictable and difficult to regulate. In addition to this, previous protein design strategies have mainly used the standard 20 amino acids as the main building blocks, which has resulted in a large unexplored potential amino acid chemical space. Although computational design methods have been adapted to incorporate non-classical amino acids into proteins for structural optimization, the technology to accurately position these non-classical amino acids at key functional sites is still in its infancy.

[0004] Genetic code expansion technology provides exceptional control over protein function by integrating non-canonical amino acids into proteins through the redistribution of codons via orthogonal aminoacyl-tRNA synthetase / tRNA pairing. 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 new functions such as light sensing and bioorthogonal chemistry into target proteins. Azobenzene, one of the classic photoswitchable groups, has a reversible, light-induced, wavelength-selective cis / trans isomerization reaction with high quantum yield. Under light with a wavelength of 340nm, the azobenzene of phenylalanine-4'-azobenzene (AzoF), a photosensitive non-natural amino acid, can photoisomerize from the more stable E-isomer to the Z-isomer under light with a wavelength of 420nm and can be restored to the E-isomer under light with a wavelength of 420nm. It has been widely used in biological environments to control protein motion by chemically linking adjacent residues or as a non-canonical amino acid through genetic engineering to control DNA binding, fluorescent protein function and enzyme activity. In previous studies, the incorporation site of AzoF was determined by a semi-rational 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 thereof.

[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 340nm wavelength light and in a polymerized state under 420nm 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-C 2 -5 dimer, the monomer of which comprises the amino acid sequence of SEQ ID No.: 1;

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

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

[0011] LRO-C 3 -37 trimer, the monomer of which comprises the amino acid sequence of SEQ ID No.: 4;

[0012] LRO-C 3-64 trimer, the monomer of which comprises the amino acid sequence of SEQ ID No.: 5;

[0013] LRO-C 4 -13 tetramer, the monomer of which comprises the amino acid sequence of SEQ ID No.: 6;

[0014] LRO-C 5 -1 pentamer, a monomer of which comprises the amino acid sequence of SEQ ID No.: 7, (ii) a heterodimer comprising an A chain and a V chain selected from the group consisting of:

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

[0016] LRD-7 heterodimer: its A chain includes the amino acid sequence of SEQ ID No.: 10, and its V chain includes 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 mutant refers to a sequence having at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity with the above-mentioned 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] LRO-C 2 The amino acid sequence of the -5 dimer monomer is SEQ ID No.: 1;

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

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

[0023] LRO-C 3 The amino acid sequence of the -37 trimer monomer is SEQ ID No.: 4;

[0024] LRO-C 3 The amino acid sequence of the -64 trimer monomer is SEQ ID No.: 5;

[0025] LRO-C 4 The amino acid sequence of the -13 tetramer monomer is SEQ ID No.: 6;

[0026] LRO-C 5 -1 pentamer monomer has an amino acid sequence of 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 a 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 and other tag sequences, 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, LRO-C 2 The amino acid sequence of the -5 dimer monomer is SEQ ID No.: 30;

[0032] LRO-C 2 The amino acid sequence of the -35 dimer monomer is SEQ ID No.: 31;

[0033] LRO-C 3 The amino acid sequence of the -7 trimer monomer is SEQ ID No.: 32;

[0034] LRO-C 3 -The amino acid sequence of the 37 trimer monomer is SEQ ID No.: 33;

[0035] LRO-C 3 The amino acid sequence of the -64 trimer monomer is SEQ ID No.: 34;

[0036] LRO-C 4 The amino acid sequence of the -13 tetramer monomer is SEQ ID No.: 35;

[0037] LRO-C 5 -1 pentamer monomer has an amino acid sequence of 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 two states of gel and solution in response to light, for example, it can present a gel state under light of 420nm wavelength and a solution state under light of 340nm wavelength.

[0043] In a specific embodiment, the homomer is the above-mentioned LRO-C 5 -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, expressing and purifying 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, a linker1, a MBP sequence, a linker2, the LRO-C 5 -1 monomer sequence; and

[0046] A plasmid for expressing a homodimer Dimer-SpyCatcher protein is constructed, and the homodimer Dimer-SpyCatcher protein is expressed and purified to obtain the homodimer Dimer-SpyCatcher protein. The monomer sequence of the homodimer 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 mass 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] Fusion protein SpyTag-MBP_LRO-C 5 -1: It contains SpyTag sequence, linker1, MBP sequence, linker2, LRO-C from N-terminus to C-terminus. 5 -1 monomer sequence; and

[0053] Homodimeric Dimer-SpyCatcher protein: Its monomer sequence includes 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, so its selection may not be limited, for example, it may be a conventional PBS or TBS buffer.

[0055] In a specific embodiment, 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 fusion protein SpyTag-MBP_LRO-C 5 -1 has an amino acid sequence of SEQ ID No.: 14, and an amino acid sequence of a homodimer Dimer-SpyCatcher protein monomer has an amino acid sequence of SEQ ID No.: 15.

[0057] In a specific embodiment, the photoresponsive hydrogel system is converted from a gel state to a liquid state under 340nm light irradiation, and returns to a gel state under 420nm 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, and 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 to promote cell secretion of reporter gene products (e.g., SEAP enzymes). Specifically, by treating the molecular sensor with 340nm light, the cell signaling pathway (e.g., JAK / STAT, PLCG, MAPK) conduction can be controlled to inhibit cell secretion of reporter gene products (e.g., SEAP enzymes), and by treating the molecular sensor with 420nm light, the cell signaling pathway (JAK / STAT, PLCG, MAPK) conduction can be activated to promote cell secretion of reporter gene products (e.g., SEAP enzymes).

[0065] Beneficial Effects

[0066] The present invention precisely designs a series of homologous 2, 3, 4, and 5-mers (respectively LRO-C 2 -5. LRO-C 2 -35, LRO-C 3 -7. LRO-C 3 -37, LRO-C 3 -64, LRO-C 4 -13. LRO-C 5 -1) and heterologous 2-mer proteins (LRD-2, LRD-7, LRD-10, respectively), 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 polymers under 340nm light and restore their aggregation state or interaction mode under 420nm light were obtained in the screening. Among them, the affinity difference of LRD-7 is nearly 177 times. In addition, the screened proteins have small molecular weight, good stability, 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 5-mers in this series of light-sensitive proteins, the present application further designed and prepared a light-sensitive protein hydrogel. Under the irradiation of light with a wavelength of 340nm, the hydrogel transforms from a gel (gel) to a solution (sol) state, and under the irradiation of light with a wavelength of 420nm, it transforms from a solution (sol) to a gel (gel). According to the test of the rotational rheometer, the storage modulus changes by nearly 10 times, and this transformation can be repeated without a significant decrease in properties.

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

[0069] Figure 1 The schematic diagram shows the design, synthesis, principle of photoresponsive chromatography and application in photoresponsive gel and extracellular molecule sensor of the light-responsive assembly protein in the present application.

[0070] Figure 2 LRO-C 2 -5. LRO-C 3 -7. LRO-C 4 -13. LRO-C 5 -1 photoresponsive gel chromatography results.

[0071] Figure 3 LRO-C 2 -5. LRO-C 3 -7. LRO-C 4 -13. LRO-C 5 -1, Model structure diagram of LRD-2, LRD-7 and LRD-10 designs.

[0072] Figure 4 LRO-C 2 -5. LRO-C 3 -7. LRO-C 4 -13. LRO-C 5 -1 analytical ultracentrifugation results.

[0073] Figure 5 LRO-C 2 -35 crystal structure under different pH conditions, and LRO-C 3 -37, LRO-C 3 -64 model structure diagram, and the light response gel chromatography results of the above three proteins. Among them, a. LRO-C 2 -35 crystal structure at pH 8.0; b. LRO-C 2 -35 crystal structure at pH 4.5; c. LRO-C 2 -35 photoresponsive gel chromatography results; d. LRO-C 3 -37 model structure diagram; e.LRO-C 3-64 model structure diagram; f.LRO-C 3 -37 photoresponsive gel chromatography results; g. LRO-C 3 -64 photoresponsive gel chromatography results.

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

[0075] Figure 7 The schematic diagram shows the construction mode of preparing light-responsive hydrogel and the light-responsive process. A represents the construction of each protein molecule in the hydrogel; B represents the change of 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] Fig. 9 The storage modulus of the photoresponsive hydrogel in two states and when switching between the two states is shown. A: Storage modulus in the gel state and in the liquid state after 340nm irradiation when the protein-solvent ratio is 10% w / v; B: Experimental results of 340 / 420nm alternating and repeated irradiation of the gel.

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

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

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

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

[0082] Fig.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] Fig.15 Shown is the crystal structure of the photoresponsive assembly protein and the verification of the electron cloud density of AzoF. DETAILED DESCRIPTION

[0084] The technical solution of the present application is 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 the same (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] Embodiment 1:

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

[0091] The designed homopolymer (LRO) includes C 2 Polymer (45), C 3 Polymer (55), C 4 Polymer (43), C 5The monomer coding sequences of the polymer (45) were synthesized between NcoI and XhoI of pET-28a by sequence synthesis (Azenta), where the codon of Azof is TAG. The synthesized plasmid was transformed into a strain containing AzoF aminoacyl-tRNA synthetase and specific tRNA by chemical transformation, incubation, heat shock, recovery, and plate coating. The strain was transformed into a conventional BL21 (DE3) (New England Biolabs) by a plasmid containing a specific AzoF aminoacyl-tRNA synthetase and tRNA AzoRS4 (ref: / / doi.org / 10.1002 / adfm.202011276), and then the competent state was prepared by the Inoue method to obtain the AzoF expression competent BL21 (DE3, AzoRS4). After bacterial growth on the plate, single clone bacteria were picked and cultured in liquid LB at 37°C, 220 rpm. After OD600 reached 0.8, 0.3 mM IPTG (Sangon Biotech) and AzoF (WuXi AppTec) unnatural amino acid at a final concentration of 0.1 mg / ml were added, and the shaking temperature was lowered to 22°C, with the speed unchanged, to express the target protein. 15 hr after induction, the bacteria were collected by centrifugation at 10000g for 5 min, and the bacteria were resuspended in about 15 ml of TBS (150 mM NaCl, 20 mM Tris pH7.4) buffer, and then ultrasonically disrupted the bacteria at 80% power for 15 min, and centrifuged at 17000g to remove the precipitate. The supernatant was purified by 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 method is also applicable to the construction of each plasmid, expression and purification of the target protein in Examples 2 and 3 below.

[0093] Analysis of the Photoresponsive Properties of Protein Aggregates

[0094] The prepared protein was divided into two parts, each part was 2 ml, one part was directly analyzed by gel chromatography for peak position, and the other part was analyzed for its depolymerization characteristics by gel chromatography after irradiation with a 340nm laser with a diameter of 1 cm in a 1.5ml centrifuge tube. The analytical column was Superdex 75increase, 10 / 300GL, the chromatograph was Yonglian Bio UEV25M, the sample volume was 1.8ml, the flow rate was 0.8ml / min, the temperature was 24°C, and the protein amount was about 0.3mg. After comprehensively judging the peak position and light responsiveness, further testing was performed to obtain the characteristics of protein recovery aggregation after 420nm irradiation.

[0095] like Figure 2 As shown in the figure, four proteins (LRO-C 2 -5. LRO-C 3 -7. LRO-C 4 -13. LRO-C 5 -1, the monomer sequences they designed were SEQ ID No.: 1, SEQ ID No.: 3, SEQ ID No.: 6, SEQ ID No.: 7). After 340nm irradiation, these four proteins were closer to the monomer peak position (15-16ml). The samples treated with 340nm irradiation with 420nm laser were able to restore their peak position in the natural state.

[0096] Further, an analytical ultracentrifuge (Beckman, Optima AUC03061703) was used at a protein concentration of about 0.4 mg / ml, 42,000 rpm, and a collection interval of 20 seconds. The resin centerpiece was used, 390 μl of the experimental group was added, and 400 μl of TBS was added to the blank control group. The sample pool 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-C 2 -5. LRO-C 3 -7. LRO-C 4 -13. LRO-C 5 -1. Their model diagram is drawn using ChimeraX, and the result is as follows Figure 3 As shown in (first row), the 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 5 As shown, LRO-C 2 -35 (whose monomer sequence is shown in SEQ ID No.: 2) has a greater impact on the aggregation state by concentration, but is less affected by 340nm light treatment. 3 -37 (whose monomer sequence is shown in SEQ ID No.: 4) tends to aggregate after 420 nm light treatment, LRO-C 3-64 (whose monomer sequence is shown in SEQ ID No.: 5) aggregated to a certain extent after both light treatments.

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

[0100] LRO-C 2 -5 dimer monomer: SEQ ID No.: 30;

[0101] LRO-C 2 -35 dimer monomer: SEQ ID No.: 31;

[0102] LRO-C 3 -7 trimer monomer: SEQ ID No.: 32;

[0103] LRO-C 3 -37 trimer monomer: SEQ ID No.: 33;

[0104] LRO-C 3 -64 trimer monomer: SEQ ID No.: 34;

[0105] LRO-C 4 -13 tetramer monomer: SEQ ID No.: 35;

[0106] LRO-C 5 -1 pentamer monomer: SEQ ID No.: 36.

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

[0108] The light-responsive pentamer LRO-C obtained in Example 1 was selected 5 -1 for the preparation of photoresponsive hydrogels.

[0109] like Figure 7 As shown, first LRO-C 5 -1 monomer sequence (its monomer sequence is shown in SEQ ID No.: 7) was added with maltose binding protein (MBP) protein at the N-terminus. On the one hand, the addition of MBP increased the protein expression, and on the other hand, increased the size of the protein and improved the modulus of gel making. The SpyTag tag was fused to the N-terminus of MBP for covalent connection to prepare hydrogel. The complete pentamer fragment was named SpyTag-MBP-LRO-C5-1 (ST-MBP_LRO-C 5)(SEQ ID No.: 14). The amino acid sequence was codon-optimized and expressed in E. coli. The optimized coding nucleic acid sequence was constructed between NcoI and XhoI of pET28a. The constructed plasmid was transformed into AzoF expression competent BL21 (DE3, AzoRS4) for expression purification.

[0110] The homologous dimer protein Dimer-SpyCatcher protein required for hydrogel preparation is selected from the dimer protein reported in the literature (C 2 -(GGS) 5 -SpyCatcher(C 2 -SC), also known as homodimeric Dimer-SpyCatcher protein, SEQ ID No.: 15).

[0111] After plasmid construction and protein purification, ST-MBP_LRO-C 5 and C 2 -SC protein, the two proteins were concentrated in TBS (150mM NaCl, 20mM Tris pH 7.4) through a 10kd ultrafiltration tube (Millipore) to a concentration of 200mg / ml. During the ultrafiltration process, the solution medium was replaced with TBS, and then ST-MBP_LRO-C 5 and C 2 -SC two proteins were mixed at a molar ratio of 1:2.5, so that ST-MBP_LRO-C 5 and C 2 The total mass of the protein in the SC accounts for 6%-20% w / v of the total mass of the solution. After about 1 hour of reaction between SpyTag and SpyCatcher, a gel is 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 the irradiation of two wavelengths of light. It transforms from the gel state to the liquid state under the 340nm light, and returns to the gel state under the subsequent 420nm light.

[0113] Storage modulus determination 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 Fig. 9As 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 repeatedly at 340 / 420 nm. Fig. 9 As shown in B, the storage modulus changes uniformly and stably under repeated switching between the gel and liquid states.

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

[0117] In this example, the screening of light-responsive heterodimers and the verification of their effects were performed.

[0118] The two parts of the designed heterodimer were constructed on the same vector for co-expression. The expressed A chain protein (LRD-2A, 7A, 10A) is the part containing AzoF and contains a 6*His tag at the tail end for affinity purification, and the V chain (LRD-2V, 7V, 10V) is the other half without AzoF. The C-terminus of the V chain is connected by a linker (GGS). 2 A monomeric GFP protein (mBaojin; PMID: 38409224) was added, wherein the amino acid sequence of the GFP protein is SEQ ID No.: 28. The addition of the GFP protein facilitates observation and increases the molecular weight difference between the A and V chains, making it easier to determine the change in the peak position under 340 nm illumination by gel chromatography.

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

[0120] The A and V chains of the three proteins were expressed separately, and an AVI tag (GLNDIFEAQKIEWHE SEQ ID No.: 29) was added to the C-terminus of the V chain for BLI testing. The BLI experiment was performed using biotinylated LRO-2V-AVI, 7V-AVI and 10V-AVI, as well as LRO-2A, 7A and 10A and AzoF site mutant proteins (AzoF amino acid site mutated to alanine or phenylalanine). The three AVI-tagged proteins purified by gel chromatography were replaced by ultrafiltration into 80mM NaCl, 20mM Tris pH 7.4, and then biotinylated using BirA-RT Kit (Avidity), with a labeled sample concentration of 100μM, reacted at 25°C for 2hr, and 5μg BirA biotinylase was added to each reaction. Subsequently, desalting and purification were performed by gel chromatography, and the protein peak was collected and concentrated to about 1mg / ml using an ultrafiltration tube. After labeling, the BLI experiment was performed using the SA probe (ForteBio) and the instrument used was OCTET RED96E (ForteBio). The concentration of the ligand bound to the probe was 10 μg / ml. The highest concentration of LRO-2A, 7A and 10A was 900 nM, and two-fold gradient dilution was performed to carry out related experiments. TBST (150 mM NaCl, 20 mM Tris pH 7.4, 0.05% Tween-20) was used to dilute the relevant proteins, and the working volume of each well of BLI was 200 μl.

[0121] like Fig.11 As shown in the figure, orthogonal experiments show that LRD-2A / 7A / 10A interact with the corresponding LRD-2V-AVI / 7V-AVI / 10V-AVI with good specificity, and even at a concentration of 900 nM, the response value of the interaction with non-corresponding proteins is very weak. The concentration gradient results of LRD-2A / 7A / 10A show 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 Fig.12 As shown in Figure 2, the 340 nm light response K of LRD-7 d The maximum reduction factor can reach 177 times, and the K of LRD-2 / 10 d The reduction factor is relatively low; the protein concentration used in the mutation experiment is 900nM. The results show that mutating the AzoF amino acid site in LRD-2A / 7A to alanine or phenylalanine by a point mutation kit (TakaRa) can eliminate its interaction with the V protein, while the two mutations of LRD-10A greatly 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 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 molecule sensor. The experimental principle is shown in Fig.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.: 16JAK / 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.: 18MAPK pathway), by inserting the coding sequence of 7V between the BamHI and EcoRI restriction sites of the above-mentioned vector; the amino acid sequences of the receptor proteins expressed by the above-mentioned plasmids are SEQ ID No.: 19, SEQ ID No.: 20 and SEQ ID No.: 21 respectively.

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

[0128] 3. The cells used were 293T cells, which were cultured in an incubator containing 10% fetal bovine serum (WISENT) and 1% streptomycin / penicillin (Thermo Fisher) at 37°C and 5% CO 2The cells were cultured in a humidified incubator. The experiment was performed in a 24-well plate, with 50,000 HEK293T cells per well. After 12 hours of cell culture, the cells were transfected. The JAK / STAT pathway was transfected with 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 according to DNA:PEI (PolyScience) 3:1 (w / w). The medium was changed 12 hours after transfection.

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

[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 report (PMID: 29686358).

[0131] like Fig.14 As shown, the final concentrations of proteins added to the three pathways of JAK / STAT3, PLCG, and MAPK were 3, 25, and 6 nM, respectively. The results showed that the SEAP enzyme activity of the untreated D7A protein (Native) was increased by 4.7, 4.8, and 20.4 times, respectively, compared with the blank group (Blank) without protein addition. The ability of D7A protein to activate cells to secrete SEAP enzymes treated with 340nm light decreased by 2.8, 2.7, and 3.3 times, respectively, and the treatment of cells at two time points could restore the activity of D7A protein to activate SEAP enzyme secretion to varying degrees.

[0132] Example 5: Crystal Screening

[0133] In this example, some proteins involved in responding to light and then assembling (including LRO-C 2 -5. LRO-C 3 -7. LRO-C 4 -13. LRO-C 5 -1, LRD-2 / 7) were screened using crystal screening kits including PEGRx (Hampton), Crystal Screen (Hampton) and Wizard Classic 1-4 (Rigaku), and XRD (Rigaku) ​​was used to pick protein crystals using loops of corresponding sizes, and the crystals were diffracted. After collecting the data, the designed model was used to analyze the structure based on molecular replacement using Phenix software. The results are as follows Fig.15 As shown in Figure 2, the obtained crystal structure is very close to the designed model, and AzoF has significant electron cloud density in all the 6 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 have a diameter of about 1cm, and the 340nm is 240mW / cm 2 , 420nm: 1500mW / cm 2 The light source was 2 cm away from the sample being treated. The light treatment time varied according to the volume. The treatment time for 1 ml of protein solution was 120 s at 340 nm and 60 s at 420 nm. The treatment time for hydrogel was similar to that for protein solution. The irradiation time was increased according to the volume of the sample.

[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 linker+6*His.

Claims

1. A light-responsive assembly protein containing phenylalanine-4'-azobenzene or a mutant thereof, which is in a disaggregated state under 340nm wavelength light and in a polymerized state under 420nm wavelength light, the light-responsive assembly protein comprising: (i) a homomer selected from the group consisting of: LRO-C2-5 dimer, whose monomer includes the amino acid sequence of SEQ ID No.: 1; LRO-C2-35 dimer, whose monomer includes the amino acid sequence of SEQ ID No.: 2; LRO-C3-7 trimer, whose monomer includes the amino acid sequence of SEQ ID No.: 3; LRO-C3-37 trimer, whose monomer includes the amino acid sequence of SEQ ID No.: 4; LRO-C3-64 trimer, whose monomer includes the amino acid sequence of SEQ ID No.: 5; LRO-C4-13 tetramer, a monomer of which comprises the amino acid sequence of SEQ ID No.: 6; LRO-C5-1 pentamer, whose monomer includes the amino acid sequence of SEQ ID No.: 7, (ii) a heterodimer comprising an A chain and a V chain selected from the group consisting of: LRD-2 heterodimer: its A chain includes the amino acid sequence of SEQ ID No.: 8, and its V chain includes the amino acid sequence of SEQ ID No.: 9; LRD-7 heterodimer: its A chain includes the amino acid sequence of SEQ ID No.: 10, and its V chain includes the amino acid sequence of SEQ ID No.: 11; 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, The mutant refers to a sequence having at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity with the above-mentioned sequences SEQ ID No.: 1-13, respectively, provided that the phenylalanine-4'-azobenzene contained in each sequence does not change.

2. The light-responsive assembly protein or a mutant thereof according to claim 1, wherein The amino acid sequence of the LRO-C2-5 dimer monomer is SEQ ID No.: 1; The amino acid sequence of the LRO-C2-35 dimer monomer is SEQ ID No.: 2; The amino acid sequence of the LRO-C3-7 trimer monomer is SEQ ID No.: 3; The amino acid sequence of the LRO-C3-37 trimer monomer is SEQ ID No.: 4; The amino acid sequence of the LRO-C3-64 trimer monomer is SEQ ID No.: 5; The amino acid sequence of the LRO-C4-13 tetramer monomer is SEQ ID No.: 6; The amino acid sequence of the LRO-C5-1 pentamer monomer is SEQ ID No.: 7; 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; 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; 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.

3. The light-responsive assembly protein or a mutant thereof according to claim 1, wherein The N-terminus of each of the above amino acid sequences also includes the sequence MG, and / or the C-terminus of each of the above amino acid sequences also includes a tag sequence for purification, such as 6*His, MBP, GST, HA, c-Myc, Flag tag sequence, 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 , Specifically, the amino acid sequence of the LRO-C2-5 dimer monomer is SEQ ID No.: 30; The amino acid sequence of the LRO-C2-35 dimer monomer is SEQ ID No.: 31; The amino acid sequence of the LRO-C3-7 trimer monomer is SEQ ID No.: 32; The amino acid sequence of the LRO-C3-37 trimer monomer is SEQ ID No.: 33; The amino acid sequence of the LRO-C3-64 trimer monomer is SEQ ID No.: 34; The amino acid sequence of the LRO-C4-13 tetramer monomer is SEQ ID No.: 35; The amino acid sequence of the LRO-C5-1 pentamer monomer is SEQ ID No.: 36; 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; 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; 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.

4. 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, a linker1, an MBP sequence, a linker2, and the LRO-C5-1 monomer sequence described above; and A plasmid for expressing a homodimer Dimer-SpyCatcher protein is constructed, and the homodimer Dimer-SpyCatcher protein is expressed and purified to obtain the homodimer Dimer-SpyCatcher protein. The monomer sequence of the homodimer 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 mass ratio in the final solution is between 6% and 20%, and a gel is formed after reaction.

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

6. The method according to claim 4, 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.

7. The method according to claim 4, wherein: 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.

8. A photoresponsive hydrogel system comprising the following two proteins in an aqueous buffer dispersion medium: Fusion protein SpyTag-MBP-LRO-C5-1: which comprises Spy-tag sequence, linker1, MBP sequence, linker2, and LRO-C5-1 monomer sequence from N-terminus to C-terminus; and Homodimer Dimer-SpyCatcher protein: Its monomer sequence contains homodimer sequence, linker3, and spy-cartcher sequence from N-terminus to C-terminus. Preferably, 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.

9. Use of the photoresponsive hydrogel system as claimed in claim 8 in preparing cell culture medium.

10. Use of the LRD-7 heterodimer as claimed in claim 1 in preparing a light-responsive extracellular molecule sensor, in, The light-responsive extracellular molecule sensor is 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. Preferably, 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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