CN117802137A - Construction and extraction method of in-situ self-polymerization hydrogel in living cells - Google Patents

Construction and extraction method of in-situ self-polymerization hydrogel in living cells Download PDF

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CN117802137A
CN117802137A CN202410182756.6A CN202410182756A CN117802137A CN 117802137 A CN117802137 A CN 117802137A CN 202410182756 A CN202410182756 A CN 202410182756A CN 117802137 A CN117802137 A CN 117802137A
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protein
elastin
dopa
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钱志刚
祝亚娇
夏小霞
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Shanghai Jiaotong University
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Abstract

The method for constructing and extracting in-situ self-polymerization hydrogel in living cells comprises the steps of constructing expression vectors of the similar leg elastin or fusion expression vectors of recombinant similar leg elastin and mussel foot protein, respectively introducing the similar leg elastin and the fusion expression vectors into tyrosine auxotroph expression hosts, and introducing the recombinant protein into the living cells to perform self-polymerization after replacing restriction substrates of tyrosine and dopa in a culture medium and raising the temperature. In the invention, dopa is introduced into typical internal disordered structural proteins in model organism escherichia coli, and intracellular in-situ self-polymerized protein hydrogel is directly obtained by utilizing various covalent and non-covalent crosslinking chemical reactions mediated by dopa residues.

Description

Construction and extraction method of in-situ self-polymerization hydrogel in living cells
Technical Field
The invention relates to a technology in the field of bioengineering, in particular to a method for constructing and extracting in-situ self-polymerization hydrogel in living cells.
Background
The biomacromolecule aggregate in the cell exists in different substance states such as liquid, gel, solid and the like, is a unique subcellular structure and plays a plurality of important roles in physiological processes such as cell viability, embryo development and the like. These subcellular compartments are thought to be driven by the liquid-liquid phase separation of the biomacromolecule and are likely to transition further to the gel state, but the structural properties and formation process of intracellular gel-like aggregates are currently unknown.
Disclosure of Invention
Aiming at the defect that the hydrogel prepared by the prior art does not have good biocompatibility and biodegradability of protein-based materials, the invention provides a construction and extraction method of an in-situ self-polymerization hydrogel in living cells, which utilizes genetic engineering and fermentation engineering technology to introduce unnatural amino acid-dopa into typical internal disordered structural proteins in model biological escherichia coli, and utilizes various covalent and non-covalent crosslinking chemical reactions mediated by dopa residues to directly obtain the in-situ self-polymerization protein hydrogel in cells, thereby not only avoiding a series of complex operation problems, but also ensuring cell viability and physiological state. The method is easy to amplify in batches, the yield of the extracted intracellular protein gel exceeds 0.67g/L (dry weight), and the extracted intracellular protein gel has good mechanical properties, and expands the construction paradigm and research range of the intracellular hydrogel.
The invention is realized by the following technical scheme:
the invention relates to a construction and extraction method of in-situ self-polymerization hydrogel in living cells, which comprises the steps of respectively introducing expression vectors of similar leg elastin or expression vectors of fusion of recombinant similar leg elastin and mussel foot protein into tyrosine auxotroph expression hosts, and introducing recombinant protein into the living cells to self-polymerize to form the in-situ hydrogel after replacement of restriction substrate tyrosine and dopa in a culture medium and temperature increase.
The arthropod elastin refers to: a protein comprising a concatamer of the conserved peptide monomers R (GGRPSDSYGAPGGGN) of Drosophila melanogaster (Drosophila melanogaster) as shown in Seq ID No.1 or a concatamer of the repeated peptide monomers Ri (GGRPSDSYGAPGGGNY) of the artificial design of the arthropod-like elastin as shown in Seq ID No. 2.
The mussel foot protein refers to: has the amino acid sequence of mussel (Mytilus edulis) podin mfp3 shown in Seq ID No.3 or mussel (Mytilus edulis) podin mfp5 shown in Seq ID No. 4.
The fusion of the recombinant meropenia elastin and mussel foot protein is as follows: fusion protein RmR of concatemer of 32 segment elastin conserved peptide segment monomers R and mussel foot protein mfp3 or mussel foot protein mfp5 block, wherein the expression plasmid vector is pTrc99A-RmR.
The amino acid sequence of the conserved peptide fragment of the arthropod elastin is derived from the literature Ardell, D.H., andersen, S.O. Infectbiochem molecular biology,2001,31,965-970.
The expression vector of the meromelanin is as follows: expression plasmid vector pTrc99A-R of 16-64 concatamers of the said leg elastin conserved peptide segment monomer R or concatamers of artificial design of leg elastin like repeated peptide segment monomer Ri N /Ri N Wherein: n represents the number of repetitions.
The tyrosine auxotroph expression host is E.coli BW25113tyrA:: kan R Knocking out tyrA gene of the tyrosine synthesis path of the escherichia coli by a homologous recombination method.
The culture medium refers to: r/2 inorganic salt synthetic medium containing antibiotics.
The replacement of the restriction substrates tyrosine and dopa refers to: the constant concentration of tyrosine and dopa is externally added into the culture medium, specifically: since the tyrosine auxotroph expression host cannot synthesize endogenous tyrosine, the tyrosine added externally in the initial culture medium is exhausted by recombinant escherichia coli, and after 2 hours of starvation, dopa and an inducer are added, wherein the final concentration of dopa is 0.5-4mM; the protein expression inducer is isopropyl thiogalactoside (IPTG) with a final concentration of 0.05-5mM.
The temperature increase means: culturing at 22-42deg.C.
The introduction is as follows: in a culture environment of 22-42 ℃, recombinant E.coli intracellular high-efficiency synthesis site-directed dopa-introduced recombinant protein, wherein dopa residues can undergo oxidation, coordination crosslinking and other reactions; when the expression is induced for 6 hours or more, dense gelatinous aggregates can be observed in living cells.
The invention relates to an intracellular self-polymerization hydrogel prepared by the method, which has a macroscopic form of a shapable viscoelastic hydrogel, a porous structure is arranged in the hydrogel, the Young modulus range is 1-40kPa, and the adhesion strength range is 1-70kPa.
The invention relates to a mass production realization method of living cell self-polymerization hydrogel prepared based on the method, which is characterized in that expression vectors of merogenesis-like elastin or fusion proteins of merogenesis-like elastin and mussel foot protein are constructed and introduced into a tyrosine auxotroph expression host, after high-density culture in a fermentation tank, the culture temperature is increased, thalli of the cell self-polymerization hydrogel are obtained, and the rapid recovery of the intracellular dopa cross-linked protein hydrogel can be realized through one-step bacterial breaking treatment.
The one-step bacteria breaking treatment means: the wet cell of Escherichia coli forming intracellular hydrogel was prepared by mixing the following components: 10 mass-bulk specific gravity is suspended in Buffer A, and then is crushed by high-pressure homogenizer to release intracellular gel; and (3) centrifuging the thallus lysate at 12000rpm for 20-30min, and washing the purple-black hydrogel in the precipitate in BufferA for 3 times to obtain the high-purity protein hydrogel.
Buffer A is an aqueous solution containing 100mM Tris, 150mM NaCl and 25mM ascorbic acid, pH7.8.
The high-purity protein hydrogel is obtained by dissolving the extracted intracellular protein hydrogel in 8M urea solution and performing SDS-PAGE analysis on the solution, wherein the main component of the extracted intracellular protein hydrogel is R32D protein, the protein purity is higher than 90%, the strips show that a large amount of dimers, multimers and the like formed by oxidative crosslinking of the protein are simultaneously present in the hydrogel except for R32D protein monomers introduced by dopa.
The yield of the extracted intracellular protein hydrogel can reach 0.67g/L (dry weight).
Technical effects
The invention synthesizes the recombinant class leg elastin introduced at the fixed point in the living cell of the escherichia coli, and crosslinks and forms gel, the class leg elastin is used as a substrate to introduce the dopa group at the fixed point, and the intracellular self-polymerization protein gel is constructed by improving the induction expression temperature; combining a high-density fermentation strategy of an expression host with one-step sterilization treatment; compared with the prior art, the invention can construct the intracellular in-situ self-polymerization hydrogel only by increasing the induction expression temperature; and the intracellular dopa crosslinked protein hydrogel can be rapidly recovered through one-step sterilization treatment without complex purification preparation flow, so that reference significance is provided for construction, extraction and research of various different types of intracellular protein gel.
Drawings
FIG. 1 is a schematic diagram of the NBT-specific stained dopa-induced protein of example 1 after SDS-PAGE of whole mycoprotein at different induction temperatures;
in the figure: w represents a whole bacterial sample, S represents a supernatant after the bacteria are broken, and P represents a sediment after the bacteria are broken;
FIG. 2 is a schematic diagram of whole-cell display E.coli intracellular gel-like aggregates of example 1;
FIG. 3 is a schematic diagram showing E.coli intracellular gel-like aggregates in sections of example 1;
FIG. 4 is a schematic representation of a gel-like aggregate in E.coli cells fluorescently labeled according to example 2;
FIG. 5 is a schematic drawing showing the extraction procedure of E.coli intracellular protein gel of example 3;
FIG. 6 is a graph showing the physical and performance properties of the intracellular R32D protein hydrogels extracted in example 3;
FIG. 7 is a schematic diagram showing the analysis of the protein component of the intracellular hydrogel extracted in example 3;
FIG. 8 is a graph showing the frequency scanning results of the intracellular R32D protein hydrogels extracted in example 4;
FIG. 9 is a graph showing the self-healing property analysis of the intracellular R32D protein hydrogels extracted in example 4;
FIG. 10 is a scanning electron micrograph of the intracellular R32D protein hydrogel extracted in example 4.
Detailed Description
Example 1
The present example relates to a method for inducing the formation of self-polymerizing protein gel-like aggregates in living cells of E.coli, comprising:
1) Construction of protein expression vector pTrc99A-R consisting of 32 concatamers of conserved peptide stretch of meropentin (amino acid sequence GGRPSDSYGAPGGGN) 32 It is introduced into a tyrosine auxotroph expression host E.coli BW25113tyrA:: kan R In (a) and (b);
2) The recombinant expression strain was inoculated into LB liquid medium containing kanamycin and ampicillin double resistance, and shake-cultured overnight at 37℃at 220 rpm. mu.L of the overnight cultured bacterial liquid is sucked into a centrifuge tube, centrifuged at 4000rpm for 5min at low temperature, and after the supernatant is removed, the bacterial cells are resuspended with an equal volume of fresh R/2 medium to exclude the interference of tyrosine in LB medium.
3) The resuspended cells were inoculated in shake flasks containing 50mL of R/2 medium (containing 25. Mu.g/mL of kanamycin, 100. Mu.g/mL of ampicillin, 20mg/L of 19 essential amino acids and 6mg/L of tyrosine) and shake-cultured at 37℃and 220 rpm. The cell density in the shake flask was measured every hour, and after the OD600 reached 0.8, the tyrosine in the medium was depleted and the cell density did not increase. Starvation culture of the cells was continued for 2 hours at 37℃and then induction culture was performed with addition of dopa and IPTG at 16 or 30℃and sampling was performed, and it was detected by SDS-PAGE that the target protein was in a completely soluble state at 16℃at low temperature and cross-linked already intracellular at 30℃as shown in FIG. 1.
The LB culture medium comprises the following components: 10g/L tryptone, 5g/L yeast powder and 10g/L sodium chloride.
The R/2 culture medium comprises the following components: 2g/L of diammonium hydrogen phosphate, 6.75g/L of monopotassium phosphate, 0.93g/L of citric acid monohydrate, 0.5% (v/v) of trace metal salt solution, 10g/L of glucose and 0.7g/L of magnesium sulfate heptahydrate.
The feed supplement liquid comprises the following components: 700g/L glucose, 20g/L magnesium sulfate heptahydrate.
4) The formation of dense protein gel-like aggregates in E.coli cells at 30℃was observed by Transmission Electron Microscopy (TEM), as shown in FIGS. 2 and 3.
And (3) preparing a sample of whole cells, re-suspending and washing the collected escherichia coli cells with deionized water for 3 times after induction, dripping 10 mu L of bacteria on a carbon film surface of a copper net, standing for 20min, slowly sucking redundant liquid with filter paper, and air-drying at room temperature.
For preparation of cell slice samples, the collected somatic cells were first resuspended in 2.5% glutaraldehyde PBS solution, fixed overnight at 4 ℃, then rinsed with 0.1M phosphate buffer (pH 7.4) and fixed with osmium acid, then dehydrated with a series of concentration gradients of ethanol and acetone, and polymerized in ethylene oxide resin at 60 ℃ for 48h. Cutting the embedded and plasticized thallus block into slices with the thickness of 80-100nm by an ultrathin slicer, transferring the slices onto a copper mesh carbon film, dyeing again by using lead citrate and uranium-free dye liquor in sequence, fully rinsing by using deionized water, and naturally air-drying a sample.
Example 2
The present embodiment relates to a method for constructing an intracellular self-polymerizing hydrogel having fluorescence activity, comprising:
1) Construction of expression vector pTrc99A-R of fusion protein of monomer concatamer of conserved peptide of 32 arthropod elastin and HaloTag structural domain 32 Halo, introduction thereof into a tyrosine auxotroph expression host E.coli BW25113tyrA:: kan R Is a kind of medium.
2) The recombinant expression strain was inoculated into LB liquid medium containing kanamycin and ampicillin double resistance, and shake-cultured overnight at 37℃at 220 rpm.
3) After replacing the medium with 500. Mu.L of seed solution, the seed solution was inoculated into a shake flask containing 50mL of R/2 medium (containing 25. Mu.g/mL of kanamycin, 100. Mu.g/mL of ampicillin, 20mg/L of 19 essential amino acids and 6mg/L of tyrosine) and shake-cultured at 37℃and 220 rpm. The cell density in the shake flask was measured every hour, and after the OD600 reached 0.8, the tyrosine in the medium was depleted and the cell density did not increase. Starvation culture of the cells was continued for 2h at 37℃followed by addition of dopa and IPTG, induction culture at 30℃for 6h, resuspension of the sampled and collected cells with PBS and addition of HaloTag ligand TMR, incubation at 30℃for 30min.
4) The bacterial cells were washed with clean PBS and the intracellular TMR-labeled protein aggregates of E.coli were observed under a fluorescence microscope as shown in FIG. 4.
The HaloTag domain is a mutated dehalogenase obtained from Rhodococcus rhodochrous and has a molecular weight of about 34kDa and can be covalently bonded to a variety of chlorinated alkane ligands, such as fluorescent molecule TMR.
The LB culture medium comprises the following components: 10g/L tryptone, 5g/L yeast powder and 10g/L sodium chloride.
The R/2 culture medium comprises the following components: 2g/L of diammonium hydrogen phosphate, 6.75g/L of monopotassium phosphate, 0.93g/L of citric acid monohydrate, 0.5% (v/v) of trace metal salt solution, 10g/L of glucose and 0.7g/L of magnesium sulfate heptahydrate.
Example 3
The present embodiment relates to a mass production and extraction method of an intracellular self-polymerizing hydrogel, comprising:
1) By constructing expression vectors pTrc99A-R of monomer concatamers of 32 segment elastin conserved peptide fragments 32 It is introduced into a tyrosine auxotroph expression host E.coli BW25113tyrA:: kan R Is a kind of medium.
2) The strain was inoculated in LB liquid medium containing kanamycin and ampicillin double resistance, cultured overnight at 37℃and transferred to R/2 medium (containing 25. Mu.g/mL of kanamycin, 100. Mu.g/mL of ampicillin, 40mg/L of 20 essential amino acids) in an inoculum size of 1%, and shake-cultured at 37℃for 7 hours to an OD600 of 2-3.
3) 10% of the inoculation amount is transferred into a 2L R/2 culture medium of a fermentation tank, and the culture medium contains 50 mug/mL of kananamycin, 100 mug/mL of ampicillin and 20mg/L of 19 essential amino acids, and tyrosine is added exogenously in batches, wherein the concentration of each addition is 100mg/L, and the total addition is 4 times. Detecting the thallus density of the fermentation liquid in each hour in the fermentation process, controlling the dissolved oxygen to be about 40%, regulating and controlling the carbon source feed by pH, culturing at 37 ℃ until the OD600 reaches to 60, continuing starving culture for 2 hours, adding dopa and IPTG, and performing induction culture at 30 ℃ for 6 hours to obtain a large number of thallus which form intracellular self-polymerization hydrogel.
4) Coli wet cells forming intracellular hydrogel were treated with 1:10 mass-bulk specific gravity was suspended in Buffer A and then broken by high pressure homogenizer 600-800bar to release intracellular gel. And (3) centrifuging the bacterial lysate after bacterial breaking at 12000rpm for 20-30min, scraping the purple-black hydrogel in the sediment, washing and centrifuging the purple-black hydrogel in BufferA, and repeating the steps for 3 times to obtain the high-purity protein hydrogel. The intracellular hydrogel extraction and washing flow is schematically shown in FIG. 5.
FIG. 6 shows the sample and performance of the intracellular hydrogel extracted in this example.
The feed supplement liquid comprises the following components: 700g/L glucose, 20g/L magnesium sulfate heptahydrate.
Buffer A is an aqueous solution containing 100mM Tris, 150mM NaCl and 25mM ascorbic acid, pH7.8.
5) The extracted intracellular protein hydrogel is dissolved in 8M urea solution, the hydrogen bond and hydrophobic interaction in the aqueous gel are broken, so that the hydrogel is partially dissolved, and SDS-PAGE analysis is carried out on the dissolution solution. The main component of the extracted intracellular hydrogel is R32D protein, the band shows that a large number of dimers, multimers and the like formed by oxidative crosslinking of the protein are simultaneously present in the hydrogel except for the R32D protein monomer introduced by dopa, the purity of the target protein is higher than 90%, and the result is shown in figure 7.
Structural and mechanical property testing
The intracellular self-polymerizing hydrogel extracted in example 3 was subjected to rheological analysis: the extracted and washed intracellular protein hydrogels were molded in a cylindrical plastic mold having a diameter of 20mm and a thickness of 1mm, and then the storage modulus and loss modulus of the hydrogels were tested using a rotational rheometer, and the gel was subjected to an oscillating shear test with frequency under a constant strain of 1%, to obtain gels, which are typical viscoelastic hydrogels, as shown in fig. 8. The hydrogel was shown to have good self-healing properties by starting to decrease the modulus value of the hydrogel under increased strain until the gel network breaks, and then again simultaneously detecting the change in modulus value over time at a constant strain of 1%, frequency of 1Hz, as shown in fig. 9.
SEM images of the hydrogels described in this example are shown in fig. 10.
Compared with the prior art that in-situ self-polymerization protein hydrogel cannot be constructed in prokaryotic cells and mass extraction cannot be realized, the method for preparing protein hydrogel by utilizing the bioengineering technology is simple and convenient in flow, and the obtained dopa crosslinked hydrogel has excellent structure and mechanical properties.
The foregoing embodiments may be partially modified in numerous ways by those skilled in the art without departing from the principles and spirit of the invention, the scope of which is defined in the claims and not by the foregoing embodiments, and all such implementations are within the scope of the invention.

Claims (10)

1. The method for constructing and extracting the in-situ self-polymerization hydrogel in living cells is characterized in that the expression vector of the similar leg elastin or the expression vector of the fusion of the recombinant similar leg elastin and mussel foot protein is respectively introduced into a tyrosine auxotroph expression host, and the in-situ hydrogel is formed by introducing the recombinant protein into the living cells for self-polymerization after the replacement of restriction substrates tyrosine and dopa in a culture medium and the temperature increase;
the arthropod elastin refers to: a protein comprising a concatemer of the conservative peptide fragment monomer R (GGRPSDSYGAPGGGN) of Drosophila melanogaster (Drosophila melanogaster) as shown in Seq ID No.1 or a concatemer of the repeated peptide fragment monomer Ri (GGRPSDSYGAPGGGNY) of the artificial design of the arthropod-like elastin as shown in Seq ID No. 2;
the mussel foot protein refers to: has the amino acid sequence of mussel (Mytilus edulis) podin mfp3 shown in Seq ID No.3 or mussel (Mytilus edulis) podin mfp5 shown in Seq ID No. 4.
2. The method for constructing and extracting in-situ self-polymerizing hydrogel in living cells according to claim 1, wherein the fusion of the recombinant meropenia elastin and mussel foot protein is: fusion protein RmR of concatemer of 32 segment elastin conserved peptide segment monomers R and mussel foot protein mfp3 or mussel foot protein mfp5 block, wherein the expression plasmid vector is pTrc99A-RmR.
3. The method for constructing and extracting in-situ self-polymerizing hydrogel in living cells according to claim 1, wherein the expression vector of the meromelanin is: expression plasmid vector pTrc99A-R of 16-64 concatamers of the said leg elastin conserved peptide segment monomer R or concatamers of artificial design of leg elastin like repeated peptide segment monomer Ri N /Ri N Wherein: n represents the number of repetitions.
4. The method for constructing and extracting in-situ self-polymerizing hydrogel in living cells according to claim 1, wherein the culture medium is: r/2 inorganic salt synthetic medium containing antibiotics.
5. The method for constructing and extracting in situ self-polymerizing hydrogel in living cells according to claim 1, wherein the replacement of restriction substrates tyrosine and dopa is: the constant concentration of tyrosine and dopa is externally added into the culture medium, specifically: since the tyrosine auxotroph expression host cannot synthesize endogenous tyrosine, the tyrosine added externally in the initial culture medium is exhausted by recombinant escherichia coli, and after 2 hours of starvation, dopa and an inducer are added, wherein the final concentration of dopa is 0.5-4mM; the protein expression inducer is isopropyl thiogalactoside (IPTG) with a final concentration of 0.05-5mM.
6. The method for constructing and extracting in-situ self-polymerizing hydrogel in living cells according to claim 1, wherein the elevated temperature is: culturing at 22-42deg.C.
7. The method for constructing and extracting in-situ self-polymerizing hydrogel in living cells according to claim 1, wherein the introducing is as follows: in a culture environment of 22-42 ℃, recombinant E.coli intracellular high-efficiency synthesis site-directed dopa-introduced recombinant protein, wherein dopa residues can undergo oxidation, coordination crosslinking and other reactions; when the expression is induced for 6 hours or more, dense gelatinous aggregates can be observed in living cells.
8. An intracellular self-polymerizing hydrogel prepared by the method of any one of claims 1 to 7, which is a moldable viscoelastic hydrogel having a porous structure therein, a young's modulus in the range of 1 to 40kPa, and an adhesion strength in the range of 1 to 70kPa.
9. A mass production realization method based on the living cell self-polymerization hydrogel of claim 8, which is characterized in that the method comprises the steps of constructing an expression vector of the meropenia-like elastin or the fusion protein of the meropenia-like elastin and mussel foot protein, introducing the expression vector into a tyrosine auxotroph expression host, culturing the expression host in a fermentation tank at high density, raising the culture temperature to obtain thalli of the cell self-polymerization hydrogel, and realizing the rapid recovery of the intracellular dopa crosslinked protein hydrogel through one-step bacterial breaking treatment.
10. The method according to claim 9, wherein the one-step sterilization treatment is: the wet cell of Escherichia coli forming intracellular hydrogel was prepared by mixing the following components: 10 mass-bulk specific gravity is suspended in Buffer A, and then is crushed by high-pressure homogenizer to release intracellular gel; after the thallus lysate is centrifuged at 12000rpm for 20-30min, the purplish black hydrogel in the sediment is taken and washed in BufferA for 3 times, thus obtaining the high-purity protein hydrogel;
buffer A was an aqueous solution containing 100mM Tris, 150mM NaCl and 25mM ascorbic acid, pH7.8.
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