CN112500495A - Purification method and application of ELP-III type collagen - Google Patents
Purification method and application of ELP-III type collagen Download PDFInfo
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- CN112500495A CN112500495A CN202011286904.7A CN202011286904A CN112500495A CN 112500495 A CN112500495 A CN 112500495A CN 202011286904 A CN202011286904 A CN 202011286904A CN 112500495 A CN112500495 A CN 112500495A
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Abstract
The invention discloses a method for purifying and renaturing fusion protein of Elastin-like polypeptides (ELPs) and collagen type III (collagen III). The invention connects the elastin-like label ELP100 and type III collagen polypeptide gene to prokaryotic expression carrier pRELPN to construct pRELPN-CLP III-ELP 100 fusion protein expression carrier, which is highly expressed in BL21(DE3) colibacillus. Since the protein exists in the form of intracellular inclusion bodies in Escherichia coli, the fusion protein is purified and renatured by adopting an inclusion body purification and renaturation method, which comprises the following steps: collecting and crushing strains, washing and removing hybrid protein, lipid and nucleic acid, purifying protein essence, renaturation by a titration dilution method, ITC (International transfer therapy) purification and MTT (methyl thiazolyl tetrazolium) cell method activity identification.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a purification method and application of ELP-III type collagen.
Background
Elastin-like polypeptides (ELPs) are genetically engineered polypeptides that are artificially modified, have elasticity, and undergo phase transition with temperature change, and are structurally formed by connecting pentapeptide repeat units in series to form a polymer (Val-Pro-Gly-Xaa-Gly, VPGXG), wherein Xaa can be any amino acid except proline, usually valine (Val, V), alanine (Ala, a), glycine (Gly, G) leucine (Leu, L) isoleucine (Ile, I), lysine (Lys, K), phenylalanine (Phe, F), histidine (His, H), and the like, and repeat unit n is usually 20-120. At present, two main synthetic methods exist, one is a chemical synthetic method for synthesizing polypeptide, and the other is a genetic recombination synthetic method. Compared with the chemical synthesis method, the gene recombination synthesis method has the following advantages: 1. the amino acid sequence, molecular weight, space folding structure and corresponding biochemical properties of the ELPs are accurately controlled through gene coding; 2. the constructed plasmid vector containing the ELPs genes can be stored in an expression strain for a long time for sustainable production; ELPs can be correctly folded into active secondary structures or active tertiary structures under the auxiliary action of a host bacterium folding mechanism, and application research can be directly carried out through purification of later soluble proteins. ELPs have one of the biggest characteristics: temperature sensitivity. At low temperatures, ELPs are soluble in water, and the polymer chains remain disordered and rather extended. When the temperature is raised to a critical temperature, which is called transition temperature (Tt) of ELPs, the ELPs are insoluble in water, and the structure of the polypeptide chains containing water is broken down and starts to aggregate, and finally precipitates out in the form of aggregates rich in ELPs, so that the process is reversible, and thus the fusion protein containing ELP tags is often purified by using Inverse Transition Cycling (ITC). The pRELPN carrier described in the patent application No. 20181003668.1 published by the company promotes independent high expression of an exogenous target gene cloned after an initial code ATG after ELP fusion protein, the fusion protein is rarely expressed, the expression of host self-protein such as escherichia coli or eukaryotic cells is slightly inhibited, and the accumulation and high expression of recombinant protein of the exogenous target gene are promoted, so that the problem that the ordinary expression carrier cannot be industrialized due to low expression or no expression of the exogenous gene or the fusion gene formed by the exogenous gene and the ELP is solved.
Type III collagen is a kind of fibroblast collagen, and has a high content in blood vessels, playing an important role. On the one hand, it can enhance the strength and elasticity of blood vessels, and on the other hand, it can provide sufficient nutrition for cells. Type III collagen also binds directly to the angioblasts, thereby forming new blood vessels. Type III collagen is an important element for keeping the skin plump, smooth and glossy. After entering human body, the type III collagen can be rapidly absorbed by the human body, and can supplement the collagen which is gradually reduced along with the increase of the age in the skin. Thereby the skin has elasticity and sufficient moisture and the fishtail lines and the raised lines of the human body can be obviously reduced. Currently, there are several main ways to produce collagen: traditional extraction method, chemical synthesis method, and modern biotechnology production method. The traditional extraction is mainly obtained from animal fur and connective tissue by an acid method, an alkali method and an enzyme method, and the obtained collagen has low activity and potential safety hazard. With the development of genetic engineering and biotechnology, since the 80 s in the 19 th century, people begin to produce collagen by using a biotechnology method and are popularized to meet the urgent needs of people on collagen, and meanwhile, the cost is saved, and greater economic benefits are brought to industrial production of collagen. There are still many problems, such as: the protein purification is complex, and needs to be purified by a series of chromatographic methods such as ion exchange, molecular exclusion and the like, and the cost is high.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: in order to solve the problems in the prior art, an improved method for purifying ELP-III type collagen and the application thereof are provided.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for purifying ELP-type iii collagen, comprising the steps of:
the first step is as follows: synthesizing an ELP100 gene sequence, constructing a pET28a-BamH-ELP100-NdeI recombinant vector, introducing the recombinant vector into DH5a escherichia coli, constructing a pRELPN-BamH-ELP100-NdeI recombinant plasmid, and introducing the recombinant plasmid into DH5a escherichia coli;
the second step is that: synthesizing type III collagen gene, constructing pRELPN-BamHI-ELP100-NdeI-CLP III-XhoI recombinant plasmid, and transfecting into escherichia coli BL21(DE3) for expression;
the third step: homogenizing and crushing escherichia coli to obtain an inclusion body containing target protein, and performing coarse purification and fine purification on the target protein by using a non-chromatographic method;
the fourth step: renaturing the inclusion body by a titration dilution method, and concentrating the renatured target protein by ultrafiltration;
the fifth step: purifying the renatured fusion protein by using Inverse Transformation Cycling (ITC);
and a sixth step: the activity of the fusion protein of ELP and type III collagen after ITC purification is detected by MTT method.
The type III collagen is composed of eight repeated segments containing 30 amino acids (CLP III), and the molecular mass of the type III collagen is 27 kDa.
The ELP is an elastin-like polypeptide formed by connecting or cloning 100 pentapeptide repeating units (Val-Pro-Gly-His-Gly, VPGHG) in series at intervals through enzyme cutting sites.
The fusion protein is composed of an ELP label and type III collagen, the middle of the fusion protein is connected through a BamHI enzyme cutting site, and the relative molecular weight of the fusion protein is 73 kDa.
The pRELPN-BamHI-ELP100-NdeI-CLP III-XhoI recombinant plasmid is constructed in the following way:
(1) the ELP100 gene was synthesized and inserted between the BamHI and NdeI cleavage sites of pET28a vector to form pET28a-BamH-ELP100-NdeI vector.
(2) A BamHI-ELP100-NdeI fragment was amplified by PCR and ligated to vector pRELPN to construct pRELPN-BamHI-ELP100-NdeI recombinant vector.
(3) The type III collagen gene is synthesized and inserted between NdeI and XhoI enzyme cutting sites of the pRELPN-BamHI-ELP100-NdeI recombinant vector to form pRELPN-BamHI-ELP100-NdeI-CLP III-XhoI recombinant plasmid.
An application of ELP-III type collagen, the ELP100-CLP III fusion protein has the function of promoting cell growth.
The invention has the beneficial effects that:
the improved ELP-III type collagen purification method and the application of the invention can be used for preparing human-like collagen on an industrial large scale, are not only suitable for the purification and renaturation of the ELP-III type collagen, but also can be used for the separation, purification and renaturation of other recombinant proteins existing in the form of inclusion bodies, and comprise the following steps: collecting and crushing strains, washing and removing hybrid protein, lipid and nucleic acid, purifying protein essence, renaturation by a titration dilution method, ITC (International transfer therapy) purification and MTT (methyl thiazolyl tetrazolium) cell method activity identification.
Drawings
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 is an electrophoretogram of purified collagen ELP-III.
FIG. 2 ELP-type III collagen renaturation and ITC purification profiles.
FIG. 3 is the rate of promotion of cell proliferation by gelatin or ELP-type III collagen fusion protein.
FIG. 4 is a comparison of cell morphology of L929 cells treated for 24h under different conditions.
FIG. 5 shows the rate of promotion of cell proliferation by various concentrations of ELP-type III collagen fusion protein after 72h of culture.
FIG. 6 is a comparison of cell morphology of L929 cells treated with different concentrations of ELP-type III collagen fusion protein for 24 hours.
Detailed Description
The present invention will now be described in further detail with reference to the accompanying drawings. These drawings are simplified schematic views illustrating only the basic structure of the present invention in a schematic manner, and thus show only the constitution related to the present invention.
The invention discloses a method for purifying improved ELP-III collagen and application thereof shown in figure 1, figure 2, figure 3, figure 4, figure 5 and figure 6, and comprises the following steps:
construction of ELP100-CLP III fusion protein expression vector
(1) The ELP100 gene was synthesized and inserted between the BamHI and NdeI cleavage sites of the vector pET28a to form a pET28a-BamH-ELP100-NdeI vector, which was introduced into DH5a E.coli.
(2) The vector pET28a was extracted and a BamHI-ELP100-NdeI fragment was amplified by PCR, ligated to the vector pRELPN to construct a pRELPN-BamHI-ELP100-NdeI recombinant vector, and introduced into DH5a E.coli.
(3) A type III collagen gene was synthesized and inserted between NdeI and XhoI cleavage sites of a pRELPN-BamHI-ELP100-NdeI recombinant vector to construct a pRELPN-BamHI-ELP100-NdeI-CLP III-XhoI recombinant plasmid.
Wherein (ELP)100 gene is constructed:
artificially synthesizing an elastin-like polypeptide (ELP) n. (VPGXG) n pentapeptide repeat units X in tandem is histidine and n is 100. Specifically, the ELP100 gene was synthesized separately in advance by a Biotech company, Inc., and inserted between BamHI and NdeI of pET28a vector to form pET28a-ELP100 vector. The sequence of type III collagen unimer amino acids is:
Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly；
ELP100 amino acid sequence Listing
Val Tyr Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Gly His Gly Val Pro Pro Arg。
Secondly, constructing a type III collagen (CLP III) gene:
based on the repetitive sequence and characteristics of the human type III collagen, a section of human type III collagen gene is designed and artificially synthesized. The front end of the DNA fragment carries NdeI and XhoI restriction enzyme sequences to form an NdeI-CLP III-XhoI fragment. The recombinant plasmid pET28a-NdeI-CLP III-XhoI was previously inserted between XhoI and NdeI of the pET28a vector.
The type III collagen gene sequence table is as follows:
GGATCCCACCACCCTCTCGCGCCTCTCGCGCCTCTCGCGCCTCGTAGGCCTCTCGCGCCTCTGGACCCTGGTGTTCCTTAACGTCCTGTTGCGCCCCACCACCCTCTCGCGCCTCTCGCGCCTCTCGCGCCTCGTAGGCCTCTCGCGCCTCTGGACCCTGGTGTTCCTTAACGTCCTGTTGCGCCCCACCACCCTCTCGCGCCTCTCGCGCCTCTCGCGCCTCGTAGGCCTCTCGCGCCTCTGGACCCTGGTGTTCCTTAACGTCCTGTTGCGCCCCACCACCCTCTCGCGCCTCTCGCGCCTCTCGCGCCTCGTAGGCCTCTCGCGCCTCTGGACCCTGGTGTTCCTTAACGTCCTGTTGCGCCCCACCACCCTCTCGCGCCTCTCGCGCCTCTCGCGCCTCGTAGGCCTCTCGCGCCTCTGGACCCTGGTGTTCCTTAACGTCCTGTTGCGCCCCACCACCCTCTCGCGCCTCTCGCGCCTCTCGCGCCTCGTAGGCCTCTCGCGCCTCTGGACCCTGGTGTTCCTTAACGTCCTGTTGCGCCCCACCACCCTCTCGCGCCTCTCGCGCCTCTCGCGCCTCGTAGGCCTCTCGCGCCTCTGGACCCTGGTGTTCCTTAACGTCCTGTTGCGCCCCACCACCCTCTCGCGCCTCTCGCGCCTCTCGCGCCTCGTAGGCCTCTCGCGCCTCTGGACCCTGGTGTTCCTTAACGTCCTGTTGCGCCCCTCGAG；
type III collagen amino acid sequence table
Pro Arg Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly
Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly
Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly
Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly
Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly
Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly
Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly
Val Val Gly Glu Arg Gly Glu Arg Gly Glu Arg Gly Ala Ser Gly Glu Arg Gly Asp Leu Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Glu Leu；
Thirdly, constructing pRELPN-BamHI-ELP100-NdeI recombinant vector:
BamHI-ELP100-NdeI fragment in pET28a-ELP100 vector was amplified by PCR. The pRELPN plasmid was cleaved with BamHI and NdeI enzymes, and the BamHI-ELP100-NdeI fragment was ligated to the cleaved pRELPN plasmid to construct a pRELPN-BamHI-ELP100-NdeI recombinant plasmid, which was introduced into DH5a E.coli.
Fourthly, constructing pRELPN-NdeI-ELP100-BamHI-CLP III-XhoI recombinant vector:
NdeI-CLP III-XhoI of pET28a-NdeI-CLP III-XhoI recombinant plasmid is amplified by PCR. Escherichia coli containing the pRELPN-BamHI-ELP100-NdeI recombinant plasmid was cultured in a large amount to purify the pRELPN-BamHI-ELP100-NdeI recombinant plasmid. NdeI, XhoI enzyme digestion and T4 ligase ligation are utilized to construct pRELPN-NdeI-ELP100-BamHI-CLP III-XhoI recombinant plasmid.
Fifthly, transfecting the pRELPN vector into BL21(DE3) escherichia coli, and culturing and inducing expression.
Secondly, the recombinant plasmid is introduced into BL21(DE3) escherichia coli, and the conditions of culture and expression are optimized
(1) The pRELPN-BamHI-ELP100-NdeI-CLP III-XhoI recombinant plasmid is transfected into an escherichia coli expression strain BL21(DE3), a positive monoclonal is screened by using kanamycin, amplified and cultured, induced and expressed by IPTG, collected, homogenized and crushed, and identified by SDS-PAGE electrophoresis.
(2) The conditions for culturing and expressing are as follows: preparing 500mL of LB culture medium, inoculating 200 μ L of bacterial liquid, adding 50 μ g/mL kanamycin solution, shaking the bacteria at 37 ℃ overnight, placing the mixture into a refrigerator at 4 ℃ for precooling for 30min when the OD600 value reaches 0.4-0.6, then culturing for 30min at 15 ℃, adding IPTG (isopropyl thiogalactoside) to 1mmol/L, and shaking the bacteria at 15 ℃ for expression for 40 h.
(3) Collecting thallus, homogenizing and crushing at 800bar for 5min, centrifuging at 13000rpm for 20min, collecting lower layer bacterial sludge to obtain inclusion body state collagen type III crude extract, and placing in a refrigerator at-20 deg.C for use.
Thirdly, the rough purification and the fine purification of the ELP100-CLP III fusion protein
(1) Crude purification
The resulting inclusion bodies were washed in suspension with 5-10 volumes of washing solution (tris 0.05M, EDTA 2mM, urea 2M, pH 8.0.0) and further at 8000rpm for 20 min. The washing was repeated 3 times. Protein extraction purity was checked by SDS-PAGE electrophoresis.
(2) Purification by refining
The roughly purified inclusion bodies were dissolved for 0.5h with 0.5 volume of a lysis solution (tris 0.05M, EDTA 5mM, guanidine hydrochloride 6M, mercaptoethanol 1%, pH 8.0), centrifuged at 12000rpm for 20min, the supernatant was retained, and the resulting precipitate was allowed to be dissolved again in the lysis solution. Protein extraction purity was checked by SDS-PAGE electrophoresis.
Renaturation of ELP100-CLP III fusion protein
The purified inclusion body protein obtained by dissolving the solution was added dropwise (2mL/min) to a renaturation solution (tris 0.05M, EDTA 2mM, urea 2M, 0.5M arginine, 4mM reduced glutathione, 0.4M oxidized glutathione, pH 8.0) stirred with a magnetic stirrer in a ratio of 1: 50. Wrapped with tinfoil and stirred overnight in dark.
After completion of the stirring, the mixture was centrifuged at 12000rpm for 20min, and the supernatant was retained to remove the precipitate. Then centrifuged and concentrated with 10KD ultrafilter tube at 8000rcf for 15 min. Finally, dialyzing in sterile PBS containing 10% glycerol for 8h to complete renaturation operation. 13000rpm, 4 ℃ for 20min, collecting soluble ELP-CLP III protein, and carrying out SDS-PAGE gel electrophoresis to verify the protein content and purity, and storing the protein at-20 ℃.
Fifthly, purifying the renatured fusion protein by using Inverse Transformation Cycling (ITC)
(1) Taking a proper amount of renatured fusion protein solution, adding NaCl with the final concentration of 1.5mol/L, and standing at 4 ℃ for 1 h;
(2) taking out the sample, centrifuging at 13000rpm for 20min, and keeping the supernatant;
(3) adding PEG2000 with final concentration of 10mg/mL into the supernatant, and water-bathing in a water bath kettle at 37 deg.C for 20 min;
(4) taking out the sample, centrifuging at 13000rpm for 20min, and keeping the precipitate;
(5) the pellet was solubilized with PBS at pH7.40, and the protein content and purity were verified by SDS-PAGE gel electrophoresis, and the protein was stored at-20 ℃.
Sixthly, the MTT method is utilized to determine the activity of the target protein after the reviving
The type III collagen is an extracellular matrix protein, has important influence on cell adhesion and cell proliferation activity, and is used for measuring the biological activity of the recombinant type III collagen through the adhesion and the proliferation activity of mouse fibroblasts (L929) and establishing an in vitro activity measuring method.
(ii) cell culture. Mouse fibroblast cells L929 were cultured in DMEM (low-sugar) medium containing 10% fetal bovine serum at 37 ℃ in a carbon dioxide incubator containing 5% CO 2. When the cells grow to 80% -90%, the cells are digested by 0.25% pancreatin, centrifuged at 1000rpm for 5min to collect the cells, washed twice by PBS, and then prepared into a cell suspension of 5X 104 cells/ml by a culture medium (containing 2.5% fetal calf serum) for later use.
And (9) paving the board. Sterile 0.5% gelatin, 0.07% gelatin and 0.07% type III collagen are prepared, a 96-well plate is taken, and the wells to be plated with cells are divided into four groups, namely an experimental group (0.07% type III collagen), a positive control group (0.5% gelatin and 0.07% gelatin) and a blank control group. Before cell spreading, 100 μ l of 0.5% gelatin, 0.07% gelatin and 0.07% type III collagen were added in advance to 96-well plates, respectively, and the blank control group was not added, and was left standing in a 37 ℃ carbon dioxide incubator for 1h to form a membrane. Removing liquid in the hole by suction, opening the cover, standing on a super clean bench for 3-5min, inoculating cell suspension with the concentration of 5 × 104/ml into a 96-well plate, wherein each well is 100 μ l, and culturing for 72h under the conditions of 37 ℃ and 5% CO 2.
③ after the culture, taking out the 96-well plate from the incubator, adding 10 mul of prepared MTT solution (5mg/mL) into each well, mixing uniformly, putting into a cell incubator and continuing to incubate for 4 h.
And fourthly, absorbing and discarding culture solution in the holes, adding 100 mu l of DMSO into each hole, slightly beating the frame of the 96-hole plate to fully dissolve the culture solution, and measuring the absorbance value at 490nm by using an enzyme-linked immunosorbent assay (ELISA) instrument.
Fifthly, calculating the cell proliferation rate (PI) according to the following formula
As shown in the figure, the ELP 100-CLPIII fusion protein has the function of promoting cell growth.
In the figure 1, A is a purified supernatant, B is a precipitate after the third washing, C is a supernatant after the third washing, D is a precipitate after the second washing, E is a supernatant after the second washing, F is a precipitate after the first washing, G is a supernatant after the first washing, H is a precipitate after crushing and centrifuging, I is a supernatant after crushing and centrifuging, and J is a Marker.
In FIG. 2, A is the supernatant of the ultrafiltrate after 1 ITC purification, B is the supernatant of the ultrafiltrate, C is the precipitate of centrifugation after renaturation, D is the supernatant of centrifugation after renaturation, E is the supernatant of renaturation, F is Marker, and G is the precipitate of fine purification.
In fig. 4 (a) is 0.5% gelatin; (b) is 0.07% type III collagen fusion protein; (c) 0.07% gelatin; (d) is a blank control.
FIG. 6 shows cells in which (a), (b), (c) and (d) are sequentially in the presence of 25, 50, 100, 200u g/mI collagen fusion protein; (e) is a blank control.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (6)
1. A method for purifying ELP-type iii collagen, comprising the steps of:
the first step is as follows: synthesizing an ELP100 gene sequence, constructing a pET28a-BamH-ELP100-NdeI recombinant vector, introducing the recombinant vector into DH5a escherichia coli, constructing a pRELPN-BamH-ELP100-NdeI recombinant plasmid, and introducing the recombinant plasmid into DH5a escherichia coli;
the second step is that: synthesizing type III collagen gene, constructing pRELPN-BamHI-ELP100-NdeI-CLP III-XhoI recombinant plasmid, and transfecting into escherichia coli BL21(DE3) for expression;
the third step: homogenizing and crushing escherichia coli to obtain an inclusion body containing target protein, and performing coarse purification and fine purification on the target protein by using a non-chromatographic method;
the fourth step: renaturing the inclusion body by a titration dilution method, and concentrating the renatured target protein by ultrafiltration;
the fifth step: purifying the renatured fusion protein by using Inverse Transformation Cycling (ITC);
and a sixth step: the activity of the fusion protein of ELP and type III collagen after ITC purification is detected by MTT method.
2. The purification method according to claim 1, wherein: the type III collagen is composed of eight repeated segments containing 30 amino acids (CLP III), and the molecular mass of the type III collagen is 27 kDa.
3. The purification method according to claim 1, wherein: the ELP is an elastin-like polypeptide formed by connecting or cloning 100 pentapeptide repeating units (Val-Pro-Gly-His-Gly, VPGHG) in series at intervals through enzyme cutting sites.
4. The purification method according to claim 1, wherein: the fusion protein is composed of an ELP label and type III collagen, the middle of the fusion protein is connected through a BamHI enzyme cutting site, and the relative molecular weight of the fusion protein is 73 kDa.
5. The purification method according to claim 1, wherein: the pRELPN-BamHI-ELP100-NdeI-CLP III-XhoI recombinant plasmid is constructed in the following way:
(1) the ELP100 gene was synthesized and inserted between the BamHI and NdeI cleavage sites of pET28a vector to form pET28a-BamH-ELP100-NdeI vector.
(2) A BamHI-ELP100-NdeI fragment was amplified by PCR and ligated to vector pRELPN to construct pRELPN-BamHI-ELP100-NdeI recombinant vector.
(3) The type III collagen gene is synthesized and inserted between NdeI and XhoI enzyme cutting sites of the pRELPN-BamHI-ELP100-NdeI recombinant vector to form pRELPN-BamHI-ELP100-NdeI-CLP III-XhoI recombinant plasmid.
6. The application of ELP-III type collagen is characterized in that the ELP100-CLP III fusion protein has the effect of promoting cell growth.
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