CN114315994B - Construction method for realizing non-denatured protein hydrogel through molecular self-assembly technology - Google Patents
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Abstract
The invention relates to a construction method for realizing non-denatured protein hydrogel by a molecular self-assembly technology, which comprises the following steps: a) And mutating glycine at the 40 th position of the Thermotoga maritima ferritin into glutamic acid/aspartic acid by a point mutation technology to obtain a ferritin mutant. b) The protein mutant is expressed and purified, and then a one-dimensional protein assembly is formed through zinc/cobalt/nickel/copper ion coordination. c) A second protein mutant was prepared by mutating serine at position 111 of the above mutant to histidine. d) And after the mutant expression is purified, a protein network structure is formed through zinc/cobalt/nickel/copper ion coordination, and the protein concentration is further improved to prepare the protein hydrogel. The protein hydrogel structure prepared by the method not only maintains the natural protein structure and maintains the bioactivity of the protein, but also has injectability and self-healing function, and has good application prospect in the food industry and the medical field.
Description
Technical Field
The invention relates to a method for preparing a non-denatured protein hydrogel material by a molecular self-assembly technology, wherein the prepared protein hydrogel can be reversibly regulated and controlled through metal ions, and has injectability and self-healing functions.
Background
Hydrogels are high molecular polymers with both solid and liquid phases, which generally form a three-dimensional network structure by means of physical or chemical crosslinking, and can be rapidly swelled in water and hold a large amount of water without dissolution. Hydrogel materials have received significant attention from scientists in recent years due to their unique porous structure, good viscoelasticity, and ductility. The hydrogel has wide application prospect in the fields of cell scaffolds, environmental engineering, equipment coating and the like. Some natural hydrogel materials, such as agarose, gelatin, methylcellulose and the like, are currently applied to agricultural production, and are used as a slow release material for slowly releasing pesticides or fertilizers and other substances, so that the utilization rate of the chemicals is improved, and the water retention rate of soil is improved; in addition, the artificially synthesized hydrogel prepared from chemical substances such as polyacrylamide, acrylic ester, polyacrylate and the like has important application in the directions of wastewater treatment, electrophoresis materials and the like.
In recent years, the preparation of hydrogels with functional active substances as materials has become a hot spot in the field of research and has shown great application potential. Hydrogels prepared from polysaccharides, proteins/polypeptides and nucleic acids can simulate the extracellular matrix environment, have important biological functions, and are favored by scientists. The protein hydrogel material has good biocompatibility or enzyme catalysis property, so that the protein hydrogel material becomes an international hot research field in the fields of functional materials, drug delivery, wound healing, tissue regeneration and the like. However, proteins themselves have complex structures and surface inhomogeneities, making the preparation of gels from proteins as starting materials very challenging.
With the continuous development of technologies such as molecular biology, protein self-assembly is a new research field developed in the last 20 years, and shows great potential. Protein self-assembly refers to a process of using proteins as building blocks, and enabling the proteins to be arranged in a self-oriented manner through covalent or non-covalent acting forces. Scientists can modify target proteins, and successfully construct zero-dimensional, one-dimensional, two-dimensional and three-dimensional protein assemblies by introducing acting forces at reasonable acting sites. In constructing these different protein dimension assemblies, the construction of one-dimensional protein assemblies is attractive. In nature, one-dimensional assemblies of proteins perform a number of biological functions: cytoskeletal formation, molecular transport, endocytosis, etc. In addition, assembly in one dimension is challenging and requires precise control over the length, width and dynamics of the assembly during the design process. By combining the current protein assembly technology and the design thought of a one-dimensional protein assembly, protein is firstly subjected to one-dimensional assembly to form protein fibers, and then the formed protein fibers are further subjected to self-assembly through protein interface design to form a three-dimensional protein network structure, so that the purpose of constructing protein hydrogel is achieved, and meanwhile, the natural structure and functions of the protein are reserved.
At present, a technology for preparing a non-denatured protein hydrogel structure in vitro by utilizing a self-assembly technology is not reported yet.
Disclosure of Invention
The invention aims to provide a method for constructing non-denatured protein hydrogel by a molecular self-assembly technology
In order to achieve the above object, the present invention provides the following technical solutions:
the method for constructing the protein hydrogel by using the molecular self-assembly technology comprises the following steps:
a) Design and preparation of protein mutants: the sequence of archaea ferritin from Thermotoga maritima is taken as a template (GenBank: AKE 30776.1), single-point mutation is carried out on the sequence by a PCR gene amplification technology, and a stop codon which is named TmFtn delta E is added after glycine at position 150. Then, the sequence of TmFtn delta E is used as a template, and single-point mutation is carried out on the sequence by a PCR gene amplification technology to mutate the glycine at the 40 th position of TmFtn delta E into glutamic acid/aspartic acid, so as to obtain a ferritin mutant named TmFtn delta E40D/E. The plasmid of the mutant was introduced into BL21 (DE 3) competence, E.coli was amplified in LB medium containing 50. Mu.g/mLAmp, and when OD600 reached about 0.8, the target protein was obtained by induction with the addition of 200. Mu.M IPTG. After 5 hours of induction, the cells are crushed by an ultrasonic crusher after the thalli are centrifuged, the ultrasonic time is 3s, the working interval is 4s, the power is 220W, and the cells are circulated for 90 times. Subsequently, the cell disruption solution was heated at 85℃for 15 minutes, and the supernatant was centrifuged to obtain a crude protein solution. The target protein was further purified by 60% ammonium sulfate precipitation and subsequently dialyzed into 25mM Tris-HCl buffer, pH 8.0. Further purification was performed using anion exchange column Mono-Q and gel filtration column Superdex 200, and the crude protein obtained in the previous step was loaded onto a column after the anion was equilibrated with buffer (50 mM Tris-HCl, pH 8.0) for at least 2 column volumes. A2-fold volume of buffer (50 mM Tris-HCl, pH 8.0) was used to wash away part of the non-filler bound heteroproteins, followed by gradient elution with 0-0.5MNaClpH 8.0 50mMTris-HCl. And (3) merging target proteins after SDS electrophoresis is verified, concentrating the target proteins, and performing gel purification. At least two column volumes of the gel filtration column were equilibrated with buffer (50 mM Tris-HCl, pH8.0,150mM NaCl), loaded and eluted at a flow rate of 0.5mL/min, and finally purity was checked by SDS-PAGE electrophoresis;
b) TmFtn.DELTA.E was added at 2mg/mL 40D/E The protein solution was dialyzed into HEPES/Mops/Tris buffer containing 0.5-2 mM zinc/cobalt/nickel/copper ions ph=7.0-9.0, at which time the ratio of protein to metal ions was 1:20, and dialyzing for 2-24h at room temperature to obtain a one-dimensional linear assembly structure with the width of about 5nm.
c) At TmFtn.DELTA.E 40D/E On the basis of the sequence of (a), serine at position 111 was mutated to histidine to give the sequence designated TmFtn.DELTA.E 40D/E/111H The ferritin mutant of (2) is separated and purified to prepare a protein solution with concentration of more than 0.5 mg/mL. The three-dimensional network structure assembly of the protein can be obtained by dialyzing in the same way into HEPES/Mops/Tris buffer containing 0.5-2 mM zinc/cobalt/nickel/copper ions with pH=7.0-9.0 and dialyzing for 2-24h at room temperature.
d) Adjusting TmFtn delta E 40D/E/111H The concentration of the protein is 10-50 mg/mL, zinc/cobalt/nickel/copper ions are slowly added into the protein solution for ten times and uniformly stirred, and finally the zinc/cobalt/nickel/copper ions are 5-20mM, so that the protein hydrogel material can be obtained.
The protein hydrogel prepared by the preparation method firstly realizes one-dimensional linear assembly of proteins through zinc/cobalt/nickel/copper ion metal coordination, the one-dimensional assemblies are not clustered, the width is about 5nm, and the protein hydrogel is an ideal structure for designing a gel network.
When the protein hydrogel is constructed, after a second metal coordination site is introduced on the basis of a one-dimensional linear assembly, the original one-dimensional linear assembly can be interwoven into a network structure, and the hydrogel material can be obtained by increasing the protein concentration.
The two metal coordination sites of the protein hydrogel are mutually noninterfere and mutually orthogonal, and the gel structure can be regulated and controlled through different metal types.
The protein hydrogel has the characteristic of metal reversible regulation, the protein can form gel through the response of metal ions, and the gel structure can be changed into a protein solution state under the action of 10-50mM EDTA or other chelating agents.
The protein hydrogel has injectability and a self-healing function, and when the shearing stress exceeds 10%, the gel becomes a solution state, and the recovery stress is 1% and the gel structure is reformed.
The prepared hydrogel is observed to be a staggered protein assembly structure through a transmission electron microscope under the condition of low concentration. The cross-sectional structure of the gel is directly observed by utilizing a freeze scanning electron microscope, a loose and porous network structure is presented, and the formed gel is relatively clear and transparent and can not flow down along the bottle wall after being placed upside down.
Compared with the prior art, the invention has the beneficial effects that:
the protein hydrogel prepared by the molecular self-assembly technology retains the natural structure and functional activity of the protein, widens the application scene, has injectability and self-healing function, and can meet the application requirements of the protein hydrogel in the industrial and medical fields.
Drawings
FIG. 1 is a schematic diagram of the construction of protein hydrogels using molecular self-assembly;
FIG. 2 is a transmission electron microscope image, a frozen scanning electron microscope image and a hydrogel image of a protein one-dimensional and network structure assembly.
Detailed Description
The invention relates to a method for preparing a non-denatured protein hydrogel material by a molecular self-assembly technology, wherein the prepared protein hydrogel can be reversibly regulated by metal ions, and has injectability and self-healing functions.
A method for preparing a non-denatured protein hydrogel material by a molecular self-assembly technology comprises the following steps:
(a) Design and preparation of protein mutants: the sequence of archaea ferritin from Thermotoga maritima is taken as a template (GenBank: AKE 30776.1), single-point mutation is carried out on the sequence by a PCR gene amplification technology, and a stop codon which is named TmFtn delta E is added after glycine at position 150. Then, the sequence of TmFtn delta E is used as a template, and single-point mutation is carried out on the sequence by a PCR gene amplification technology to mutate the glycine at the 40 th position of TmFtn delta E into glutamic acid/aspartic acid, so as to obtain a ferritin mutant named TmFtn delta E40D/E. The plasmid of the mutant was introduced into BL21 (DE 3) competence, E.coli was amplified in LB medium containing 50. Mu.g/mLAmp, and when OD600 reached about 0.8, the target protein was obtained by induction with the addition of 200. Mu.M IPTG. After 5 hours of induction, the cells are crushed by an ultrasonic crusher after the thalli are centrifuged, the ultrasonic time is 3s, the working interval is 4s, the power is 220W, and the cells are circulated for 90 times. Subsequently, the cell disruption solution was heated at 85℃for 15 minutes, and the supernatant was centrifuged to obtain a crude protein solution. The target protein was further purified by 60% ammonium sulfate precipitation and subsequently dialyzed into 25mM Tris-HCl buffer pH 8.0. Further purification was performed using anion exchange column Mono-Q and gel filtration column Superdex 200, and the crude protein obtained in the previous step was loaded onto a column after the anion was equilibrated with buffer (50 mM Tris-HCl, pH 8.0) for at least 2 column volumes. A2-fold volume of buffer (50 mM Tris-HCl, pH 8.0) was used to wash away part of the non-filler bound heteroproteins and then a gradient elution was performed with 0-0.5M NaCl pH8.0 50mMTris-HCl. And (3) merging target proteins after SDS electrophoresis is verified, concentrating the target proteins, and performing gel purification. Firstly, balancing at least two column volumes of a gel filtration column by using a buffer solution (50 mM Tris-HCl, pH8.0,150mM NaCl), loading and eluting, wherein the flow rate is 0.5mL/min, and finally, checking the purity by SDS-PAGE electrophoresis;
(b) TmFtn.DELTA.E was added at 2mg/mL 40D/E The protein solution was dialyzed into HEPES/Mops/Tris buffer containing 0.5-2 mM zinc/cobalt/nickel/copper ions ph=7.0-9.0, at which time the ratio of protein to metal ions was 1:20, and dialyzing for 2-24h at room temperature to obtain a one-dimensional linear assembly structure with the width of about 5nm.
(c) At TmFtn△E 40D/E On the basis of the sequence of (a), serine at position 111 was mutated to histidine to give the sequence designated TmFtn.DELTA.E 40D/E/111H The ferritin mutant of (2) is separated and purified to prepare a protein solution with concentration of more than 0.5 mg/mL. The three-dimensional network structure assembly of the protein can be obtained by dialyzing in the same way into HEPES/Mops/Tris buffer containing 0.5-2 mM zinc/cobalt/nickel/copper ions with pH=7.0-9.0 and dialyzing for 2-24h at room temperature.
(d) Adjusting TmFtn delta E 40D/E/111H The concentration of the protein is 10-50 mg/mL, zinc/cobalt/nickel/copper ions are slowly added into the protein solution for ten times and uniformly stirred, and finally the zinc/cobalt/nickel/copper ions are 5-20mM, so that the protein hydrogel material can be obtained.
In the present invention, the sequence information includes:
TmFtn△E
MMVISEKVRKALNDQLNREIYSSYLYLSMATYFDAEGFKGFAHWMKKQAQEELTHAMKFYEYIYERGGRVELEAIEKPPSNWNGIKDAFEAALKHEEFVTQSIYNILELASEEKDHATVSFLKWFVDEQVEEEDQVREILDLLEKANG(SEQ ID NO:1)
TmFtn△E 40D/E
MMVISEKVRKALNDQLNREIYSSYLYLSMATYFDAEGFKD(E)FAHWMKKQAQEELTHAMKFYEYIYERGGRVELEAIEKPPSNWNGIKDAFEAALKHEEFVTQSIYNILELASEEKDHATVSFLKWFVDEQVEEEDQVREILDLLEKANG(SEQ ID NO:2)
TmFtn△E 40D/E/111H
MMVISEKVRKALNDQLNREIYSSYLYLSMATYFDAEGFKD(E)FAHWMKKQAQEELTHAMKFYEYIYERGGRVELEAIEKPPSNWNGIKDAFEAALKHEEFVTQSIYNILELAHEEKDHATVSFLKWFVDEQVEEEDQVREILDLLEKANG(SEQ ID NO:3)
the protein hydrogel prepared by the preparation method firstly realizes one-dimensional linear assembly of proteins through zinc/cobalt/nickel/copper ion metal coordination, the one-dimensional assemblies are not clustered, the width is about 5nm, and the protein hydrogel is an ideal structure for designing a gel network.
When the protein hydrogel is constructed, after a second metal coordination site is introduced on the basis of a one-dimensional linear assembly, the original one-dimensional linear assembly can be interwoven into a network structure, and the hydrogel material can be obtained by increasing the protein concentration.
The two metal coordination sites of the protein hydrogel are mutually noninterfere and mutually orthogonal, and the gel structure can be regulated and controlled through different metal types.
The protein hydrogel has the characteristic of metal reversible regulation, the protein can form gel through the response of metal ions, and the gel structure can be changed into a protein solution state under the action of 10-50mM EDTA or other chelating agents.
The protein hydrogel has injectability and a self-healing function, and when the shearing stress exceeds 10%, the gel becomes a solution state, and the recovery stress is 1% and the gel structure is reformed.
The prepared hydrogel is observed to be a staggered protein assembly structure through a transmission electron microscope under the condition of low concentration. The cross-sectional structure of the gel is directly observed by utilizing a freeze scanning electron microscope, a loose and porous network structure is presented, and the formed gel is relatively clear and transparent and can not flow down along the bottle wall after being placed upside down.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
example 1
Protein expression and purification steps of Thermotoga maritima ferritin mutants in the present invention.
(1) Plasmid preparation and protein expression of Thermotoga maritima ferritin mutant
Design and preparation of protein mutants: the sequence of archaea ferritin from Thermotoga maritima is taken as a template (GenBank: AKE 30776.1), single-point mutation is carried out on the sequence by a PCR gene amplification technology, and a stop codon which is named TmFtn delta E is added after glycine at position 150. Then, the sequence of TmFtn delta E is used as a template, and single-point mutation is carried out on the sequence by a PCR gene amplification technology to mutate the glycine at the 40 th position of TmFtn delta E into glutamic acid/aspartic acid, so as to obtain a ferritin mutant named TmFtn delta E40D/E. Plasmids of this mutant were introduced into BL21 (DE 3) competence, and the procedure was performed by adding 5. Mu.L of the mutant plasmid to 100. Mu.L of competent cells, standing on ice for 30min, then precisely heat-shocking at 42℃in a metal bath for 60s, and then standing on ice for 2min. Subsequently, 500. Mu.L of LB medium was added to competent cells, and after resuscitating for 1 hour in a shaking table at 37℃the mixture was spread evenly on a plate containing Amp-resistant solid medium and placed in an incubator at 37 ℃. After overnight incubation, single colonies were picked up and resuscitated in 5mL vials containing a final concentration of 50. Mu.g/mLAMP, followed by inoculation into 500mL Erlenmeyer flasks for amplified incubation, and when OD600 reached about 0.8, the target protein was obtained by induction with the addition of 200. Mu.M IPTG.
(2) Plasmid preparation and protein expression of Thermotoga maritima ferritin mutant
After the protein is induced for 5 hours, the cells are crushed by an ultrasonic crusher after the thalli are centrifuged, the ultrasonic time is 3s, the working interval is 4s, the power is 220W, and the cells are circulated for 90 times. The cell disruption solution was then heated at 85℃for 15min and centrifuged at 10000rpm for 10min, followed by taking the supernatant to obtain a crude protein solution. The target protein was further precipitated by 60% ammonium sulfate, and after standing for 4h, the protein precipitate was obtained by centrifugation at 10000rpm for 10min, and then dialyzed into 25mM Tris-HCl buffer, pH8.0, for 5h each time and repeated three times. Centrifuging at 10000rpm after dialysis for 10min to obtain protein supernatant, purifying with anion exchange column Mono-Q and gel filtration column Superdex 200, balancing anions with buffer (50 mM Tris-HCl, pH 8.0) for at least 2 column volumes, and loading the crude protein obtained in the previous step. A2-fold volume of buffer (50 mM Tris-HCl, pH 8.0) was used to wash away part of the non-filler bound heteroproteins and then a gradient elution was performed with 0-0.5M NaCl pH8.0 50mM Tris-HCl. After SDS electrophoresis was performed, the target proteins were pooled, concentrated using a 10kDa cut-off concentration tube, and then gel purified. Firstly, balancing at least two column volumes of a gel filtration column by using a buffer (50 mM Tris-HCl, pH8.0,150mMNaCl), loading and eluting, wherein the flow rate is 0.5mL/min, and finally, checking the purity by SDS-PAGE electrophoresis;
example 2
The invention discloses the construction of a maritime thermophila one-dimensional protein assembly, the formation of a protein network structure and the preparation of a protein coagulant.
(1) Construction of one-dimensional and network structure protein assembly of Thermotoga maritima
TmFtn delta E of Thermotoga maritima ferritin 40D/E After separation and purification, the solution was dialyzed into a buffer (pH 8.0,25mM HEPES,250mM NaCl) containing 10 to 50mM EDTA to completely remove the free metal ions. The protein solution was then dialyzed again into buffer (ph 8.0,25mM HEPES,250mMNaCl) to remove EDTA and its metal complexes formed. Protein concentration was measured by Lowery method to prepare a protein mother liquor with a final concentration of 1 mg/mL. A zinc/cobalt/nickel/copper ion solution with a mother liquor concentration of 100mM was prepared with acidified water, diluted by the corresponding factor and added to a buffer solution at pH8.0,25mM HEPES,250mM NaCl to give a final concentration of zinc/cobalt/nickel/copper ion of 0.5mM. Dialyzing the prepared protein mother solution into a buffer solution containing zinc/cobalt/nickel/copper ions, and dialyzing for 4 hours to obtain the one-dimensional linear assembly.
The preparation of the protein network structure assembly was consistent with the above method, with TmFtn delta 40D/E/111H The preparation is successful by repeating the above operations for the raw materials.
(2) Construction of protein hydrogel and reversible regulation thereof
a. By TmFtn 40D/E/111H The mutant is used as an experimental raw material, and ultrafiltration concentration tube with the molecular weight cut-off of 100kDa is used for concentrating protein, so that protein mother liquor with the protein concentration of 10mg/mL is finally obtained. 1mL of protein mother liquor is taken and added into a transparent glass sample bottle, and a rotor is arranged in the transparent glass sample bottle for slow stirring. The acidified water was used to prepare a zinc/cobalt/nickel/copper ion solution with a mother liquor concentration of 100mM, 5. Mu.L of each time was added dropwise to the sample bottle using a pipette, and after ten minutes of sufficient stirring, 5. Mu.L of zinc/cobalt/nickel/copper ion mother liquor was continuously added dropwise, and the dropwise addition was repeated 8 times, so that the final concentration of zinc/cobalt/nickel/copper ions in the sample bottle was 4mM. The ratio of protein to zinc/cobalt/nickel/copper ions was about 1:10, the gel structure has been formed in this state as evidenced by inversion of the vial.
b. 1mL of EDTA solution with pH of 8.0 and 10-50mM is mixed with the gel in equal volume ratio, and the gel is gradually dissolved after slowly shaking for about 2min to form a solution state. The solution was dialyzed into buffer (pH 8.0,25mM HEPES,250mM NaCl) to remove EDTA and the remaining metal ions. Readjusting the protein concentration to 10mg/mL, repeating the operation of step a, and forming the gel structure again.
Example 3
(1) One-dimensional and network structure protein assembly of calcium ion induced Thermotoga maritima
TmFtn delta E of Thermotoga maritima ferritin 40D/E After isolation and purification, the solution was dialyzed into a buffer containing 10-50mM EDTA (pH 8.0,25mM HEPES,250mM NaCl) to completely remove the free metal ions. The protein solution was then dialyzed again into buffer (pH 8.0,25mM HEPES,250mMNaCl) to remove EDTA and its metal complexes formed. Protein concentration was measured by Lowery method to prepare a protein mother liquor with a final concentration of 1 mg/mL. A1M stock solution of calcium ions was prepared with acidified water, diluted by the corresponding factor and added to a buffer solution at pH8.0,25mM HEPES,250mM NaCl to give a final concentration of calcium ions of 50mM. The prepared protein mother solution is dialyzed into a buffer solution containing calcium ions, and no one-dimensional assembly is formed after dialysis for 2-24 hours.
The preparation of the calcium ion-induced protein network structure assembly was consistent with the above method, with TmFtn delta 40D/E/111H Repeating the above operations for the raw materials cannot prepare a protein network structure.
(2) Construction and reversible regulation of calcium ion pair protein hydrogel
By TmFtn 40D/E/111H The mutant is used as an experimental raw material, and ultrafiltration concentration tube with the molecular weight cut-off of 100kDa is used for concentrating protein, so that protein mother liquor with the protein concentration of 10mg/mL is finally obtained. 1mL of protein mother liquor is taken and added into a transparent glass sample bottle, and a rotor is arranged in the transparent glass sample bottle for slow stirring. The acidified water is used for preparing a calcium ion solution with the concentration of 1M, 5 mu L of the mother solution of the calcium ion is dripped into a sample bottle each time by using a liquid transferring gun, the mixture is fully stirred for ten minutes, 5 mu L of the mother solution of the calcium ion is continuously dripped, and the dripping is repeated for 10 times, so that the final concentration of the calcium ion in the sample bottle is 50mM. The inability to form a gel structure in this state was demonstrated by inverting the vial.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Chinese university of agriculture
<120> a method for constructing a non-denatured protein hydrogel by molecular self-assembly technique
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Claims (7)
1. The method for constructing the non-denatured protein hydrogel by a molecular self-assembly technology is characterized by comprising the following steps of:
a) Design and preparation of protein mutants: the sequence of GenBank accession AKE30776.1 of archaea ferritin from Thermotoga maritima is taken as a template, single-point mutation is carried out on the sequence by a PCR gene amplification technology, a stop codon is added after glycine at 150 th position, the sequence is named TmFtn delta E, then the sequence of TmFtn delta E is taken as the template, and single-point mutation is carried out on the sequence by the PCR gene amplification technology, so that glycine at 40 th position of TmFtn delta E is mutated into glutamic acid/aspartic acid, and the sequence is named TmFtn delta E 40D/E Ferritin mutants of (2) at TmFtn delta E 40D/E On the basis of the sequence of (a), serine at position 111 was mutated to histidine to give the sequence designated TmFtn.DELTA.E 40D/E/111H Ferritin mutants of (a);
b) Respectively introducing plasmids of the mutant into BL21 competence, amplifying escherichia coli in LB culture medium containing 50 mug/mL of ampicillin, when OD600 reaches about 0.8, obtaining target protein by adding 200 mug of IPTG induction, centrifuging thalli after induction for 5 hours, crushing cells by an ultrasonic crusher for 3s at a working interval of 4s and a power of 220W, circulating for 90 times, heating cell crushing liquid at 85-90 ℃ for 15-30 min, centrifuging, and obtaining a crude protein solution;
c) Purifying the crude protein solution by 30-60% ammonium sulfate precipitation, dialyzing into 25mM Tris-HCl buffer solution with pH of 8.0, balancing gel filtration column with Mono-Q and gel filtration column Superdex 200, balancing anions with pH of 8.0 and 50mM Tris-HCl buffer solution for at least 2 column volumes, loading the crude protein obtained in the previous step onto column, washing with 2 times volume of pH8.0 and 50mM Tris-HCl buffer solution to remove part of impurity proteins not combined with filler, gradient eluting with 0-1.0M NaCl pH8.0 50mM Tris-HCl, performing SDS electrophoresis to verify, combining target protein, concentrating the target protein, performing gel purification, balancing gel filtration column with pH of 8.0 and 50mM Tris-HCl and 150mM NaCl buffer solution, loading and eluting, measuring purity by SDS-PAGE, and obtaining electrophoretically pure TmFtn delta E 40D/E And TmFtn delta E 40D/E/111H A ferritin mutant;
d) TmFtn.DELTA.E at 2mg/mL 40D/E The protein solution was dialyzed into HEPES/Mops/Tris buffer containing 0.5-2 mM zinc/cobalt/nickel/copper ions ph=7.0-9.0, at which time the ratio of protein to metal ions was 1:20, dialyzing for 2-24h at room temperature to obtain a one-dimensional linear assembly structure with the width of about 5 nm;
e) TmFtn delta E of 0.5mg/mL or more 40D/E/111H The protein solution is dialyzed for 2 to 24 hours by adopting HEPES/Mops/Tris buffer solution containing 0.5 to 2mM zinc/cobalt/nickel/copper ions with pH=7.0 to 9.0 at room temperature, so that a three-dimensional network structure assembly of non-denatured protein can be obtained; tmFtn.DELTA.E 40D/E/111H The concentration of the protein is regulated to 10-50 mg/mL, zinc/cobalt/nickel/copper ions are slowly added into the protein solution for ten times and evenly stirredAnd stirring to obtain the final zinc/cobalt/nickel/copper ion of 5-20mM, thereby obtaining the protein hydrogel material.
2. The construction method according to claim 1, wherein: the one-dimensional linear assembly of the protein is realized firstly through the coordination of zinc/cobalt/nickel/copper ion metal, and the one-dimensional assembly body is not converged into clusters, and the width is 5nm.
3. The construction method according to claim 1, wherein: after a second metal coordination site is introduced on the basis of the one-dimensional linear assembly, the original one-dimensional linear assembly can be interwoven into a network structure, and the hydrogel material can be obtained by increasing the protein concentration.
4. The construction method according to claim 1, wherein: the two metal coordination sites are mutually noninterfere and are mutually orthogonal, and the gel structure can be regulated and controlled through different metal types.
5. The construction method according to claim 1, wherein: the hydrogel has the characteristic of metal reversible regulation, the protein can form gel through the response of metal ions, and the gel structure can be changed into a protein solution state through the action of 10-50mM EDTA or other chelating agents.
6. The construction method according to claim 1, wherein: the hydrogel has injectability and self-healing function, when the shearing stress exceeds 10%, the gel becomes a solution state, the recovery stress is 1%, and the gel structure is reformed.
7. The construction method according to claim 1, wherein: the prepared hydrogel has a staggered protein assembly structure observed by a transmission electron microscope under the condition of low concentration, and a cross-section structure is directly observed by a freeze scanning electron microscope, so that a loose and porous network structure is presented, the formed gel is relatively clear and transparent, and the formed gel cannot flow down along the bottle wall after being placed upside down.
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