CN115724916B - Elastin-like polypeptide (VPTGIG)25Preparation method and application thereof - Google Patents
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Abstract
The invention discloses an elastin-like polypeptide (VPTGIG) 25, and a preparation method and application thereof, and belongs to the technical field of biology. Firstly, synthesizing an elastin-like polypeptide (VPTGIG) 25 gene sequence, inserting the elastin-like polypeptide into a plasmid to construct a recombinant plasmid, introducing the recombinant plasmid into DH5a escherichia coli, and culturing and then extracting the recombinant plasmid; then the obtained recombinant plasmid is transfected into escherichia coli BL21 (DE 3) to induce expression, the obtained escherichia coli is crushed to obtain a soluble protein solution containing target protein, and the soluble protein solution is purified by a reversible phase-change circulation method to obtain the elastin-like polypeptide (VPTGIG) 25. The purification method of the elastin-like polypeptide (VPTGIG) 25 is simple, can realize large-scale separation and purification of elastin-like, enriches the variety of ELPs and provides a new type for drug carriers.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an elastin-like polypeptide (VPTGIG) 25, and a preparation method and application thereof.
Background
The discovery and application of novel nanomaterials with biocompatibility and self-assembly characteristics broadens the development of nanomaterials in biomedical field research. Advances in this area are expected to advance biomedical research and provide new inspiration for the design of intelligent nano-biocompatible materials with self-assembly capabilities. The newly designed materials have the characteristics of high biocompatibility, good stability, excellent pharmacokinetics and the like. Among these novel nanomaterials, elastin-like polypeptides (elastin-like polypeptides, ELPs) and their derivatives and other biocompatible materials show great potential. Elastin is an important extracellular matrix protein, which is found mainly in connective tissue of arteries, skin, lungs and ligaments, and can combine with microfibrils in the body to form elastic fibers, which together with collagen fibers impart elasticity and tensile capacity to tissues. ELPs are repetitive artificial polypeptides derived from repetitive amino acid sequences in the hydrophobic domain of tropoelastin, whose structure is mainly formed by tandem pentapeptide (Val-Pro-Gly-Xaa-Gly, VPGXG) repeats, where Xaa represents any amino acid other than proline. The most typical properties of ELPs are temperature sensitivity and self-assembly, and as the ambient temperature changes, the ELPs undergo a reversible phase change process. Under certain environmental conditions, ELPs are highly soluble if the ambient temperature is below the phase transition temperature (transition temperature, tt), whereas above that temperature they assemble to form a coacervate phase, which returns to the solution state as the temperature decreases. This phase change or self-assembly process is mainly temperature-induced aggregation, often with varying degrees of thermal hysteresis. By virtue of the special properties, the nano engineering ELPs are considered as a novel and promising drug delivery and controlled release material, and have the characteristics of prolonged drug half-life, lower drug toxicity, higher drug solubility, good biocompatibility, excellent pharmacokinetic behavior and the like. However, their possible application areas are mainly dependent on their intrinsic properties, and their further application in the biomedical field is still very limited due to their single high Tt value, uncontrolled self-assembly behaviour and unregulated hysteresis characteristics.
ELPs have important theoretical research and practical application values, but also have some problems, mainly: (1) The protein is purified in a complex way, and a series of chromatographic methods such as ion exchange, molecular exclusion and the like are needed for purification, so that the cost is high; (2) ELPs have relatively limited phase change protein sequences and relatively limited biomedical applications.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an elastin-like polypeptide (VPTGIG) 25, and a preparation method and application thereof.
The technical scheme provided by the invention is as follows:
An elastin-like polypeptide having the amino acid sequence (VPTGIG) n and n=5-50.
Based on the technical scheme, further, the amino acid sequence of the elastin-like polypeptide is (VPTGIG) 25, the Tt value at the concentration of 0.5mg/ml is 37 ℃, and the reversible phase change property is maintained.
In another aspect, the present invention provides a method for preparing the elastin-like polypeptide, which mainly comprises the following steps:
(1) Artificially synthesizing a gene sequence for encoding the elastin-like polypeptide, inserting the gene sequence into a plasmid to construct a recombinant plasmid, introducing the recombinant plasmid into DH5a escherichia coli, and culturing to extract the recombinant plasmid;
(2) Transfecting the recombinant plasmid obtained in the step (1) into escherichia coli BL21 (DE 3) to induce expression;
(3) Crushing the escherichia coli obtained in the step (2) to obtain a soluble protein solution containing target proteins;
(4) The elastin-like polypeptide is obtained by purification through a reversible phase-change circulation method.
Based on the above technical scheme, further, the plasmid in the step (1) is pET28a, and the gene fragment is inserted between NcoI and XhoI of the pET28a vector.
Based on the technical scheme, further, the gene sequence of the elastin-like polypeptide coded in the step (1) is shown as SEQ ID NO. 1.
Based on the technical scheme, further, the specific process of inducing expression in the step (2) is as follows: when the OD 600 value of the bacterial liquid reaches 0.6-0.8, 0.5-5 mmol/L IPTG is added, and the bacterial liquid is shaken at 30-37 ℃ for 3-8 h.
Based on the technical scheme, the specific mode of the crushing treatment in the step (3) is one or a combination of more than two of ultrasonic crushing, homogenizing crushing, ball milling crushing, repeated freeze thawing crushing and lysozyme enzymolysis crushing.
Based on the above technical scheme, further, the specific process of obtaining the soluble protein solution containing the target protein in the step (3) is as follows: centrifuging the E.coli crushing liquid obtained by crushing treatment at 10000-15000 rpm for 5-20 min at 1-4 ℃, discarding the precipitate, transferring the supernatant into a new centrifuge tube, repeating the steps until the centrifugation has no macroscopic precipitate, and reserving the supernatant to obtain the soluble protein solution containing the target protein.
Based on the technical scheme, the reversible phase-change circulation method in the step (4) further comprises the following steps:
1) Adding NaCl solid into the soluble protein solution containing target protein to a final concentration of 1-3 mol/L, carrying out water bath at 38-45 ℃ for 10-30 min, centrifuging at 8000-12000 rpm for 5-20 min at 30-36 ℃, and discarding the supernatant;
2) Re-suspending the precipitate obtained in the step 1) by using 0.01-0.1 mol/L PBS buffer solution, placing on ice for 5-30 min, centrifuging for 5-20 min at 8000-12000 under the condition of 1-4 ℃, discarding the precipitate, and transferring the supernatant into a new centrifuge tube.
Based on the technical scheme, the reversible phase-change circulation method circulates for more than 2 times.
The invention also provides application of the elastin-like polypeptide in preparing a drug delivery system.
Compared with the prior art, the invention has the following beneficial effects:
1. The purification method of the elastin-like polypeptide (VPTGIG) 25 is simple, reduces the preparation cost, and can realize large-scale separation and purification of elastin-like;
2. The elastin-like polypeptide (VPTGIG) 25 enriches the types of ELPs and provides a new type for drug carriers;
3. The invention explores the influence of 1,6 hexanediol on the self-assembly mechanism of the elastin-like polypeptide ELP25, is helpful for further understanding the behaviors of other ELPs which are reported before and lack the deep understanding of molecules, is helpful for designing new ELPs meeting different requirements, and expands the practical application range of the ELPs in a plurality of fields.
Drawings
In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described.
FIG. 1 is a diagram showing the cleavage assay of elastin-like polypeptide (VPTGIG) 25.
FIG. 2 is a graph of the ITC purification results for elastin-like polypeptide (VPTGIG) 25.
FIG. 3 is a graph of thermal behavior analysis of elastin-like polypeptide (VPTGIG) 25.
FIG. 4 is a graph of circular dichroism analysis of elastin-like polypeptide (VPTGIG) 25.
FIG. 5 is a graph showing the turbidity effect of varying concentrations of 1,6 hexanediol on elastin-like polypeptide (VPTGIG) 25.
Detailed Description
The following detailed description of the invention is provided in connection with examples, but the implementation of the invention is not limited thereto, and it is obvious that the examples described below are only some examples of the invention, and that it is within the scope of protection of the invention to those skilled in the art to obtain other similar examples without inventive faculty.
Example 1
Elastin-like polypeptides are mainly composed of Val-Pro-Gly-Xaa-Gly (VPGXG) n pentapeptide repeats, wherein Xaa (X) refers to amino acids other than proline (Pro), typically 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, typically the first 3 and last 1 amino acids in VPGXG remain unchanged throughout the repeat sequence where the pentapeptide repeats form n 20-120, and we change the strategy to design elastin-like polypeptides (VPTGIG, 25).
The application provides a preparation method of elastin-like polypeptide ELP25[ (VPTGIG) 25 ], which comprises the following steps:
(1) Artificially synthesizing a gene fragment of the elastin-like polypeptide ELP25 (the nucleotide sequence is shown as SEQ ID NO. 1), and amplifying the gene fragment under the specific conditions that: pre-denaturation at 95℃for 5min, denaturation at 95℃for 25s, annealing at 56℃for 30s, extension at 72℃for 25s, circulation for 30 times, extension at 72℃for 10min, and heat preservation at 10 ℃.
(2) Inserting the obtained ELP25 gene fragment (the nucleotide sequence is shown as SEQ ID NO. 1) between NcoI and XhoI of the pET28a vector to form a pET28a-ELP25 vector; the specific process is as follows: purifying the obtained ELP25 gene fragment by NcoI and XhoI double digestion and kit, connecting according to TIANGEL MIDI purification instruction book of Takara company to obtain pET28a-ELP25 vector, after enzyme digestion identification is positive, introducing pET28a-ELP25 vector into DH5 alpha competence, culturing kanamycin resistance flat plate at 37 ℃, picking up monoclonal colony, shaking at 37 ℃ overnight in 10mL common LB culture solution containing kanamycin with final concentration of 50 mug/mL;
(3) After positive identification by double digestion of NcoI and XhoI, plasmids are extracted, pET28a expression vector plasmids pET28a-ELP25 containing exogenous target genes ELP25 are transduced into escherichia coli expression bacteria BL21 (DE 3) to be competent, monoclonal bacteria are selected, 10mL of ordinary LB culture solution containing 50 mug/mL of kanamycin with final concentration is added into a 50mL test tube, shaking is carried out at 37 ℃ overnight, 100mL of ordinary LB culture solution containing 50 mug/mL of kanamycin with final concentration is added into a 300mL triangular flask, 1mL of bacterial solution is added, shaking is carried out for 3 hours at 37 ℃, when OD 600 value reaches 0.6-0.8, 1mmol/L of IPTG is added, and shaking expression is carried out at 37 ℃ for 5 hours. The cells were collected by centrifugation at 10000rpm for 10 min. Then, 30mL of 0.02mol/L PBS buffer solution was added, the mixture was sonicated, centrifuged at 17000rmp for 40min to obtain a supernatant, the supernatant was mixed with Loading Buffer carrying mercaptoethanol, and then boiled at 100℃for 5 min, centrifuged, and SDS-PAGE was performed to identify the expression molecular weight and the expression amount of the recombinant protein of (VPTGIG) 25 in the supernatant of the expressing strain.
Example 2
Purification of (VPTGIG) 25:
the corresponding purified protein can be obtained by repeating temperature-controlled reversible phase change (ITC) for 3 times, and the specific steps are as follows:
(1) Repeatedly freezing and thawing bacterial liquid stored at-80deg.C at 37 deg.C and-20deg.C for 2 times to dissolve bacterial cells completely;
(2) Adding lysozyme to make its final concentration be 50ug/mL, and making water bath at 40 deg.C for 30min to make lysozyme act;
(3) After ice is placed for a period of time, the ice bath is broken for 10min (the condition is that the ultrasonic treatment is carried out for 2s, the ultrasonic treatment is stopped for 3s, and the power is 15 percent);
(4) Placing on ice for 15min, centrifuging at 12000rpm in a low-temperature centrifuge at 4deg.C for 10min, discarding precipitate, and transferring supernatant into a new tube;
(5) Adding Dnase to make its final concentration be 50ug/mL, and making ice bath action for 20min;
(6) Centrifuging at 12000rpm in a low-temperature centrifuge at 4deg.C for 10min, discarding the precipitate, and transferring the supernatant into a new tube;
(7) Repeating the steps for a plurality of times until no obvious precipitate exists in the centrifugation;
(8) Adding NaCl solid into the supernatant to a final concentration of 2mol/L, performing water bath at 40 ℃ for at least 20min (until NaCl is completely dissolved, the bath time of the protein is relatively reduced, and the protein is subjected to pure water bath), centrifuging at 34 ℃ for 10min at 12000rpm, and discarding the supernatant;
(9) 20mL of 0.02mol/L PBS was resuspended in sediment, ice placed for 15min, centrifuged at 12000rpm in a low temperature centrifuge at 4℃for 10min, the sediment was discarded and the supernatant was transferred into a new tube;
(10) Adding NaCl solid into the supernatant to a final concentration of 2mol/L, carrying out water bath at 40 ℃ for 15min, centrifuging at 32-34 ℃ for 10min at 10000rpm, and discarding the supernatant;
(11) 20mL of 0.02mol/L PBS was resuspended in sediment, ice placed for 15min, centrifuged at 12000rpm in a low temperature centrifuge at 4℃for 10min, the sediment was discarded and the supernatant was transferred into a new tube;
(12) Adding NaCl solid into the supernatant to a final concentration of 2mol/L, carrying out water bath at 40 ℃ for 10min, centrifuging at 32-34 ℃ for 10min at 10000rp, and discarding the supernatant;
(13) 2-5 mL of 0.02mol/L PBS is subjected to resuspension precipitation, ice placement is carried out for 15min, centrifugation is carried out at 10000rpm in a low-temperature centrifuge at 4 ℃ for 8min, supernatant is separated and transferred into a new centrifuge tube for preservation, and then the soluble elastin-like polypeptide ELP25 is obtained, the protein content and purity are verified by SDS-PAGE gel electrophoresis, and the result is shown in figure 2, and the protein is preserved at-20 ℃.
Example 3
The cuvette containing the purified aqueous solution (0.5 mg/ml) of the soluble elastin-like polypeptide ELP25 of example 2 was placed in the instrument and the instrument power switch was turned on in sequence, followed by computer-window 7 operation interface and the nanoparticle size and zeta potential operating software Zetasizer software.
(1) A size is selected among the function options in the menu bar, after which a start button on the right side of point Manual measurement (which button displays green after the instrument is normally on-line) appears.
(2) Selecting size under a Measure type item in a method setting interface; inputting a Sample name under a Sample item, and selecting water under a dispersing solvent DISPERSANT item; the Temperature item inputs the set Temperature: 25 ℃; inputting the sample under Equilibration time to determine the stabilization time before measurement; the Measurement item is entered with the corresponding Number of runs, run time (Run duration) and Number of loops (Number of measurements).
(3) After the parameter is set, the measurement is started at the start point, after the measurement is finished, the main menu window is returned to find the corresponding sample file name under the records view item, and the average diameter of the result Z-average is checked under the items such as INTENSITY PSD.
(4) Finally, heating and cooling at a rate of 1 ℃/min on a ZS 90-type nanoparticle and Zeta potential analyzer equipped with a multi-unit thermoelectric temperature controller resulted in an elastin-like polypeptide (VPTGIG) 25 having a Tt value of 37 ℃ at a concentration of 0.5mg/ml and maintained reversible phase change properties as shown in fig. 3.
Example 4
CD spectroscopic detection of the soluble elastin-like polypeptide ELP25, comprising the steps of:
(1) Starting up
(2) Spectral condition setting
① The slit width was set to 0.5nm.
② Click on the upper "Acquisition Setup" of the window,
The Scanning Setup dialog box is popped up, and "absorpance" is selected in the Acquisition mode.
Corresponding test conditions are set similar to CD spectral scanning, and then "OK" is clicked on to confirm.
(3) Reference spectrum test (high voltage regulation)
Clicking on "record" of "REFERENCE SPECTRUM" records the light source spectrum.
In this process, "HV on" is on and "Auto" is off. After one reference spectrum scan is completed, the range of voltage values on the vertical axis of the spectrum is checked. The value of HV is manually input, and the reference spectrum is scanned again, and the maximum value of the vertical axis voltage of the spectrogram is suggested to be about 8V-9V. After the reference spectrum test is completed, "REFERENCE SPECTRUM" turns red.
(4) Blank test
A cuvette containing deionized water was placed in the sample chamber and a Blank was tested by clicking the "Record" button of the Blank select.
After the Blank test is completed, "Blank select" turns red. The "sub" is checked, and the blank is directly subtracted in the next sample test.
(5) Sample testing
The blank sample cuvette was removed and replaced with an aqueous solution (0.01 mg/ml) of the soluble elastin-like polypeptide ELP25, and the sample was tested by clicking the start button at Acquisition control, the results of which are shown in FIG. 4.
(6) Shutdown
Example 5
Split charging the separated and purified elastin-like polypeptide ELP25 into 6 1.5mL centrifuge tubes, and adding 0M, 1M, 2M, 3M, 4M and 5M 1,6 hexanediol aqueous solution respectively to a final elastin-like polypeptide ELP25 concentration of 0.1mg/mL. Then put it into a water bath kettle with 63 ℃ (higher than Tt value) for water bath for 5 minutes, and take out to observe the state. Solutions of the elastin-like polypeptide ELP25 at different concentrations of 1,6 hexanediol were each added in 200ul to 96-well plates and their turbidity was measured at 63℃using a UV spectrophotometer with temperature control. The results are shown in FIG. 5, and it can be seen that as the concentration of 1, 6-hexanediol increases, the turbidity of the ELPs decreases until it is completely dissolved, indicating that the main force to maintain the ELPs structure is hydrophobic.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (9)
1. An elastin-like polypeptide, wherein the elastin-like polypeptide has the amino acid sequence of (VPTGIG) 25.
2. The method for producing an elastin-like polypeptide of claim 1, comprising the steps of:
(1) Synthesizing a gene sequence for encoding the elastin-like polypeptide, inserting the gene sequence into a plasmid to construct a recombinant plasmid, introducing the recombinant plasmid into DH5a escherichia coli, and culturing to extract the recombinant plasmid;
(2) Transfecting the recombinant plasmid obtained in the step (1) into escherichia coli BL21 (DE 3) to induce expression;
(3) Crushing the escherichia coli obtained in the step (2) to obtain a soluble protein solution containing target proteins;
(4) The elastin-like polypeptide is obtained by purification through a reversible phase-change circulation method.
3. The method of claim 2, wherein the plasmid in step (1) is pET28a and the gene fragment is inserted between NcoI and XhoI of pET28a vector.
4. The method of claim 2, wherein the specific process of inducing expression in step (2) is: when the OD 600 value of the bacterial liquid reaches 0.6-0.8, 0.5-5 mmol/L IPTG is added, and the bacterial liquid is shaken at 30-37 ℃ for 3-8 h.
5. The method according to claim 2, wherein the specific manner of the crushing treatment in the step (3) is one or a combination of two or more of ultrasonic crushing, homogenizing crushing, ball milling crushing, repeated freeze thawing crushing and lysozyme enzymatic crushing.
6. The method according to claim 2, wherein the specific process for obtaining the soluble protein solution containing the target protein in the step (3) is as follows: centrifuging the E.coli crushing liquid obtained by crushing treatment at 10000-15000 rpm for 5-20 min at 1-4 ℃, discarding the precipitate, transferring the supernatant into a new centrifuge tube, repeating the steps until the centrifugation has no macroscopic precipitate, and reserving the supernatant to obtain the soluble protein solution containing the target protein.
7. The method according to any one of claims 3 to 6, wherein the reversible phase-change cycling method in step (4) comprises the steps of:
1) Adding NaCl solid into the soluble protein solution containing target protein to a final concentration of 1-3 mol/L, carrying out water bath at 38-45 ℃ for 10-30 min, centrifuging at 8000-12000 rpm for 5-20 min at 30-36 ℃, and discarding the supernatant;
2) Re-suspending the precipitate obtained in the step 1) by using 0.01-0.1 mol/L PBS buffer solution, placing on ice for 5-30 min, centrifuging for 5-20 min at 8000-12000 under the condition of 1-4 ℃, discarding the precipitate, and transferring the supernatant into a new centrifuge tube.
8. The method of claim 7, wherein the reversible phase-change cycling method is performed more than 2 times.
9. Use of an elastin-like polypeptide according to claim 1 for the preparation of a drug delivery system.
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| CN106222189A (en) * | 2016-09-06 | 2016-12-14 | 大连大学 | A kind of method based on class elastin laminin label preparation restructuring N akrencephalon pro-BNP |
| CN112500495A (en) * | 2020-11-17 | 2021-03-16 | 点斗基因科技(南京)有限公司 | Purification method and application of ELP-III type collagen |
| CN112661856A (en) * | 2020-08-20 | 2021-04-16 | 点斗基因科技(南京)有限公司 | Purification method and application of ELP collagen |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106222189A (en) * | 2016-09-06 | 2016-12-14 | 大连大学 | A kind of method based on class elastin laminin label preparation restructuring N akrencephalon pro-BNP |
| CN112661856A (en) * | 2020-08-20 | 2021-04-16 | 点斗基因科技(南京)有限公司 | Purification method and application of ELP collagen |
| CN112500495A (en) * | 2020-11-17 | 2021-03-16 | 点斗基因科技(南京)有限公司 | Purification method and application of ELP-III type collagen |
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| 类弹性蛋白多肽的从头设计、非色谱纯化及盐效应;黄凯宗;李晶晶;李巍;葛慧华;王文研;张光亚;;生物工程学报;20110425(第04期);全文 * |
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