阅读说明：本技术 一种由单一蛋白形成的智能水凝胶 (Intelligent hydrogel formed by single protein ) 是由 曾安平 张心怡 任杰 聂晶磊 于 2020-06-04 设计创作，主要内容包括：本发明属于生物材料领域,具体涉及一种由单一蛋白质形成的具有催化活力的智能性水凝胶。所述智能水凝胶是将来源于大肠杆菌的硫辛酸连接酶蛋白(LplA)稀释于缓冲溶液中,通过控制体系的理化条件形成的。所述智能水凝胶具有温度敏感性和可逆性,临界温度范围为8℃-80℃,高于临界温度时形成水凝胶,降温时回复溶液状态,并且该可逆转变过程可重复30次以上,同时具有温度响应灵敏,转变温度范围在2℃以内,温度响应时间短(3min内)的优点。所述水凝胶还可以通过调节pH和离子浓度控制凝胶状态。可广泛以用于三维细胞培养、药物缓释、靶向治疗药物制备、美容护肤品及温度传感等组织工程及医学美容领域中。(The invention belongs to the field of biological materials, and particularly relates to an intelligent hydrogel which is formed by a single protein and has catalytic activity. The intelligent hydrogel is formed by diluting lipoic acid ligase protein (LpIA) derived from escherichia coli into a buffer solution and controlling the physicochemical conditions of a system. The intelligent hydrogel has temperature sensitivity and reversibility, the critical temperature range is 8-80 ℃, the hydrogel is formed when the critical temperature is higher than the critical temperature, the solution state is recovered when the temperature is reduced, the reversible transformation process can be repeated for more than 30 times, and the intelligent hydrogel has the advantages of sensitive temperature response, transformation temperature range within 2 ℃ and short temperature response time (within 3 min). The hydrogel can also control the gel state by adjusting the pH and ion concentration. Can be widely used in the tissue engineering and medical cosmetology fields of three-dimensional cell culture, drug sustained release, targeted therapy drug preparation, beauty skin care products, temperature sensing and the like.)

1. The intelligent protein hydrogel is characterized in that lipoic acid ligase protein shown in a sequence table SEQ ID NO.1 is diluted in a buffer solution, and the intelligent protein hydrogel is formed by controlling the physicochemical conditions of a system.

2. The intelligent protein hydrogel according to claim 1, wherein said physicochemical conditions are temperature, pH or buffer ion concentration.

3. The intelligent protein hydrogel according to claim 2, wherein the critical temperature ranges from 8 ℃ to 80 ℃, and the hydrogel is formed when the critical temperature is higher than the critical temperature, and returns to a solution state when the temperature is reduced.

4. The smart hydrogel of claim 3, wherein the critical temperature is in the range of 10 ℃ to 50 ℃.

5. The protein smart hydrogel of claim 2, wherein the gel-forming pH, at which the hydrogel is formed, is in the range of 2 to 12.

6. The smart protein hydrogel of claim 5, wherein said gelling pH is in the range of 4.2 to 9.

7. The protein smart hydrogel of claim 2, wherein the gel-forming ion concentration is in the range of 5-500 mM.

8. The protein smart hydrogel of claim 7, wherein the gel-forming ion concentration is in the range of 50-100 mM.

9. The intelligent protein hydrogel of claim 1, wherein the protein concentration is 0.01-5 mM.

10. The protein smart hydrogel of claim 9, wherein the protein concentration is 0.05-2 mM.

11. The protein smart hydrogel of claim 1, wherein said buffer solution comprises but is not limited to one of Tris-HCl buffer solution, phosphate buffer solution, citrate buffer solution, or acetate buffer solution.

12. The protein intelligent hydrogel according to claim 1, wherein the lipoic acid ligase protein has a gene sequence shown as SEQ ID No. 2; alternatively, the first and second electrodes may be,

the protein is lipoic acid ligase protein encoded by a gene with homology of more than 80 percent with a sequence shown in SEQ ID No. 2; alternatively, the first and second electrodes may be,

the protein is a protein encoded by adding nucleotides with certain length at two ends of a sequence shown in SEQ ID No. 2.

13. The protein smart hydrogel of claim 12, wherein said added nucleotides are 6 histidine sequences, glutathione mercaptotransferase tag protein sequences, or maltose binding protein tag sequences.

14. The protein smart hydrogel according to claim 1, wherein said lipoic acid ligase protein is an amino acid sequence affecting protein dimer formation resulting from mutation based on SEQ ID No. 1.

15. The protein smart hydrogel of claim 14, wherein said lipoic acid ligase protein is a protein having at least one of the following mutations based on SEQ ID No. 1: S252A, H267A, D269A or Q279A.

16. The intelligent protein hydrogel as claimed in claim 1 is applied to the fields of three-dimensional cell culture, drug slow release, targeted therapy drug preparation, beauty skin care products, temperature sensing tissue engineering and medical beauty.

The technical field is as follows:

the invention belongs to the field of biological materials, and particularly relates to an intelligent hydrogel with catalytic activity, which is formed by a single protein.

Background art:

the hydrogel is a dispersion system with water as a dispersion medium in the gaps of a three-dimensional network structure. Smart hydrogels refer to a class of special hydrogels that change morphology from solution to gel as a function of environmental factors such as temperature, solution characteristics, ion concentration, and pH. Generally, smart hydrogels are transformed in response to external physical or chemical stimuli.

Among numerous intelligent hydrogels, the temperature-sensitive intelligent hydrogel is not introduced with other substances, and only has the characteristics of temperature change, simplicity, easiness in operation, environmental friendliness and the like, so that the temperature-sensitive intelligent hydrogel is concerned. The intelligent temperature-sensitive hydrogel is a special hydrogel with the shape reversibly changed between a solution and a gel along with the temperature change, and has strong application prospects in cell tissue culture, drug slow release, targeted therapy, beauty and skin care products and temperature sensing.

The intelligent temperature-sensitive hydrogel is reported to be mainly divided into natural hydrogel and artificial hydrogel, wherein the natural temperature-sensitive hydrogel mainly comprises chitosan, cellulose, hyaluronic acid and xylan, the sources of the natural temperature-sensitive hydrogel are rich but all polymers and mixtures, a single and definite molecular structure does not exist, and modification is difficult. The artificially synthesized temperature-sensitive hydrogel comprises polyethylene glycol, polyacrylamide, polyoxyethylene and the like, but has the defects of slow response speed and poor biocompatibility, and the block polymerization reaction process required for synthesizing the target molecule has relatively high requirements, relatively poor stability, environmental friendliness and high application cost. Recently, researchers have developed artificially synthesized temperature-sensitive hydrogels based on polypeptides, proteins and ribonucleic acids (DNAs), which have good biocompatibility. However, no intelligent temperature-sensitive hydrogel formed by proteins with biocatalytic activity is reported at present.

The invention content is as follows:

the invention relates to an intelligent hydrogel based on natural protein, which has simple preparation process, is environment-friendly, is easy to stably prepare in large quantities, and can change the gel property through protein engineering.

The natural protein is lipoic acid ligase protein (LplA) derived from escherichia coli, the amino acid sequence of the natural protein is shown as SEQ ID No.1, and the gene sequence of the natural protein is shown as SEQ ID No. 2; alternatively, the first and second electrodes may be,

the protein is lipoic acid ligase protein which has homology of more than 80 percent with a sequence shown in SEQ ID No.2, further has homology of more than 90 percent, and further has homology of more than 99 percent; alternatively, the first and second electrodes may be,

the protein is a protein which is encoded by adding nucleotides with certain length at two ends of a sequence shown in SEQ ID No. 2;

the added nucleotides include, but are not limited to, 6 histidine sequences (His-tag), glutathione mercaptotransferase tag protein sequences (GST-tag), or maltose binding protein tag sequences (MBP-tag);

the protein can also be a mutant of the lipoic acid ligase protein (LplA) shown in SEQ ID No.1, and the mutation generated by the mutant influences the formation of protein dimers;

further, the mutant is a protein which generates at least one of the following mutation sites on the basis of SEQ ID NO. 1: s252, H267, D269, R277, Q279, G248, P251, C57, C303 or C313;

further, the mutant is a protein which generates at least one of the following mutation sites on the basis of SEQ ID NO. 1: S252A, H267A, D269A or Q279A.

The intelligent hydrogel is formed by diluting lipoic acid ligase protein (LpIA) in a buffer solution and controlling the physicochemical conditions of a system, wherein the physicochemical conditions are temperature, pH or buffer solution ion concentration; the formation of hydrogen bonds between proteins is influenced by controlling the temperature, pH or buffer solution ion concentration of the system, so that the formation of protein dimers is influenced, and the system is converted between a gel state and a solution state;

furthermore, the intelligent hydrogel is formed by controlling the temperature, the critical temperature range is 8-80 ℃, the intelligent hydrogel is formed when the critical temperature is higher than the critical temperature, and the intelligent hydrogel returns to the solution state when being cooled; more closely, the critical temperature range is 10-50 ℃;

further, the intelligent hydrogel is formed by controlling the pH, the gelling pH range is 2-12, and the hydrogel is formed in the pH range; further, the pH range of the gel is 4.2-9;

further, the intelligent hydrogel is formed by controlling the ion concentration, the gel-forming ion concentration range is 5-500mM, and the hydrogel is formed in the ion concentration range; more particularly, the concentration of the colloid-forming ions ranges from 50 to 100 mM.

The LpIA concentration is 0.01-5mM, and the preferred concentration is 0.05-2 mM;

the buffer solution includes but is not limited to Tris-HCl buffer solution, phosphate buffer solution, citrate buffer solution and acetate buffer solution;

the invention also provides the application of the intelligent hydrogel in the tissue engineering and medical cosmetology fields of three-dimensional cell culture, drug slow release, targeted therapy drug preparation, beauty skin care products, temperature sensing and the like.

Has the advantages that:

the invention reports a reversible Temperature-sensitive hydrogel composed of single protein for the first time, which has a Low Critical Solution Temperature (LCST), shows a free-flowing sol state at a low Temperature, can be quickly converted into a gel state when the Temperature is increased to a certain Temperature, and can be quickly restored to the Solution state when the Temperature is reduced. Compared with the traditional temperature-sensitive hydrogel of natural chitosan, cellulose and the like, the hydrogel disclosed by the invention is composed of single protein, has a definite molecular structure, and is easy to modify. Compared with the artificially synthesized temperature-sensitive hydrogel such as polyethylene glycol, polyacrylamide and the like, the hydrogel disclosed by the invention is prepared by a pure biological means, so that the high-temperature and high-pressure conditions of chemical polymerization reaction are avoided, the use and the residue of an organic solvent are avoided, the hydrogel is green and environment-friendly, the biological safety is high, and the preparation process is simple and mature.

The hydrogel has the advantages of sensitive temperature response, short temperature response time (within 3 min) and capability of completing the conversion from a solution state to the gel within 2 ℃.

The hydrogel has temperature reversibility, the temperature is increased to form a gel state, the temperature is reduced to become a solution state, and the reversible conversion process can be repeated for more than 30 times.

In addition, the hydrogel disclosed by the invention also has pH sensitivity and ion concentration sensitivity, and the gel state can be adjusted by temperature, pH and the ion concentration of a buffer solution respectively.

Description of the drawings:

FIG. 1 rheological Properties of LpIA (1mM) hydrogel;

FIG. 2 gel profiles of LpIA in different pH and different buffer solutions;

FIG. 3 gelling of LpIA and 5 mutants at ambient temperature;

FIG. 4 is a schematic diagram of hydrogen bond formation between LpIA proteins.

The specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.

The lipoic acid ligase protein (LplA) can be obtained by artificial synthesis of amino acid sequences, and can also be obtained by constructing recombinant bacteria by means of gene engineering for expression, separation and purification, and an expression host comprises but is not limited to Escherichia coli BL21(DE 3); expression vectors include, but are not limited to, plasmids pET28a, pET22b, pET21a, and the like; restriction enzymes include, but are not limited to, NcoI and XhoI, etc., and the following examples will exemplify protein expression and preparation and use by genetic engineering means.

In the invention, the protein for forming gel can be lipoic acid ligase protein shown in SEQ ID NO.1, or can be protein which has certain homology with the coding gene of the lipoic acid ligase protein shown in SEQ ID NO.1, such as protein coded by nucleotide sequence which has more than 80% homology with the sequence shown in SEQ ID NO.2, further has more than 90% homology, and further has more than 99% homology; or, a protein encoded by adding a certain length of nucleotide to both ends of the sequence shown in SEQ ID No.2, preferably, the added nucleotide includes but is not limited to 6 histidine sequences (His-tag), glutathione mercaptotransferase tag protein sequences (GST-tag), or maltose binding protein tag sequences (MBP-tag); or, a protein mutated in the sequence of SEQ ID NO.1, which mutation may affect protein dimer formation, e.g., the mutation at the following positions: s252, H267, D269, R277, Q279, G248, P251, C57, C303 or C313, preferably the mutant is a protein that produces at least one of the following mutation sites on the basis of SEQ ID No. 1: S252A, H267A, D269A or Q279A.

The amino acid sequence shown in SEQ ID NO.1 is as follows:

MGSTLRLLISDSYDPWFNLAVEECIFRQMPATQRVLFLWRNADTVVIGRAQNPWKECNTRRMEEDNVRLARRSSGGGAVFHDLGNTCFTFMAGKPEYDKTISTSIVLNALNALGVSAEASGRNDLVVKTVEGDRKVSGSAYRETKDRGFHHGTLLLNADLSRLANYLNPDKKKLAAKGITSVRSRVTNLTELLPGITHEQVCEAITEAFFAHYGERVEAEIISPNKTPDLPNFAETFARQSSWEWNFGQAPAFSHLLDERFTWGGVELHFDVEKGHITRAQVFTDSLNPAPLEALAGRLQGCLYRADMLQQECEALLVDFPEQEKELRELSAWMAGAVR

in the invention, the temperature, the pH and the ion concentration of the buffer solution can respectively influence the formation of LpIA hydrogel, LpIA is dissolved in Tris-HCl buffer solution, phosphate buffer solution, citrate buffer solution or acetate buffer solution, and the formation of hydrogen bonds between proteins is influenced by controlling the temperature, the pH or the ion concentration of the buffer solution of the system, so that the formation of protein dimers is influenced, and the system is converted between the gel state and the solution state.

Wherein LplA protein solutions with different physicochemical conditions correspond to different critical temperatures, the critical temperature is between 10 and 80 ℃, preferably between 10 and 50 ℃, the solution becomes gel when the temperature is higher than the critical temperature, and the solution returns to the state of the solution when the temperature is lower than the critical temperature. Such as: in a system with pH of 5.0-8.0 and ion concentration of 100-500mM, the critical transition temperature range of the gel state is 60-80 ℃ if the concentration of LpIA is 0.01-0.05mM, the critical transition temperature range of the gel state is 40-60 ℃ if the concentration of LpIA is 0.05-0.10mM, the critical transition temperature range of the gel state is 23-40 ℃ if the concentration of LpIA is 0.10-0.30mM, the critical transition temperature range of the gel state is 13-23 ℃ if the concentration of LpIA is 0.3-1.0mM, and the critical transition temperature of the gel state is 8-13 ℃ if the concentration of LpIA is 1-4 mM;

wherein LpA protein solutions with different physicochemical conditions correspond to different gelling pH values between 2 and 12, preferably between 4.2 and 9.0. For example, in a system with a temperature of 20-50 ℃ and an ion concentration of 100-500mM, if the concentration of LpIA is 0.1-0.6mM, the pH range of the gel is 5.0-8.0, if the concentration of LpIA is 0.6-1mM, the pH range of the gel is 4.5-8.5, if the concentration of LpIA is 1-2mM, the pH range of the gel is 4.2-9.0;

wherein LplA protein solutions with different physicochemical conditions correspond to different gelling ion concentrations, and the gelling ion concentration is between 5 and 500 mM. For example, in a system with a temperature of 20-50 ℃ and a pH of 5.0-8.0, the gel forming ion concentration range is 100-500mM when the concentration of LpIA is 0.1-0.6 mM; when the concentration of LpIA is 0.6-1mM, the concentration range of colloid-forming ions is 50-500 mM; when the concentration of LpIA is 1-2mM, the concentration range of colloid-forming ions is 5-500 mM.

The buffer solution used in the present invention includes, but is not limited to, Tris-HCl buffer solution, phosphate buffer solution, citrate buffer solution or acetate buffer solution.

In the present invention, some preferred conditions for forming the gel are also included, such as:

forming a gel state when the pH of the buffer solution is about 5.0-8.0 at the temperature of 13 ℃ and the concentration of LpIA is 1 mM; the pH value is about 8.5-10, and the solution state is obtained;

forming a gel state when the pH of the buffer solution is about 5.0-8.5 at the temperature of 20 ℃ and the concentration of LpIA is 1 mM; the pH value is about 9.0-10, and the solution state is obtained;

forming a gel state when the pH of the buffer solution is about 5.0-9.0 at the temperature of 38 ℃ and the concentration of LpIA is 1 mM; the pH value is about 9.5-10, and the solution state is obtained;

forming a gel state when the pH of the buffer solution is about 5.0-8.5 at the conditions that the LpIA concentration is 0.6mM and the temperature is 38 ℃; the pH value is about 9.0-10.0, and the solution state is obtained;

under the conditions that the LpIA concentration is 1mM, the temperature is 13 ℃, and the pH value is 7.5, the buffer solution forms a solution state when the ion concentration of the buffer solution is 0.1-50mM, and forms a gel state when the ion concentration of the buffer solution is 100-500 mM;

under the conditions that the LpIA concentration is 1mM, the temperature is 38 ℃, and the pH value is 7.5, the buffer solution forms a solution state when the ion concentration of the buffer solution is 0.1-5mM, and forms a gel state when the ion concentration of the buffer solution is 10-500 mM;

under the conditions that the LpIA concentration is 0.6mM, the temperature is 20 ℃, and the pH value is 7.5, the buffer solution forms a solution state when the ion concentration of the buffer solution is 0.1-50mM, and forms a gel state when the ion concentration of the buffer solution is 100-500 mM;

under conditions of a LpIA concentration of 0.6mM, a temperature of 38 ℃ and a pH of 7.5, the buffer ion concentration is in a solution state at 0.1-5mM and in a gel state at 10-500 mM.

The invention will be further explained below by means of specific examples.