CN110484990B - Rare earth protein fiber and preparation method thereof - Google Patents
Rare earth protein fiber and preparation method thereof Download PDFInfo
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- CN110484990B CN110484990B CN201910778135.3A CN201910778135A CN110484990B CN 110484990 B CN110484990 B CN 110484990B CN 201910778135 A CN201910778135 A CN 201910778135A CN 110484990 B CN110484990 B CN 110484990B
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
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Abstract
The invention provides a rare earth protein fiber, which is prepared by drawing super positive charge protein and anionic surfactant; the anionic surfactant has a catechol building block. The super positive charge protein and the anionic surfactant form a protein-surfactant compound under the electrostatic action, and different metal ions are doped to be chelated with the protein-surfactant compound for coordination crosslinking, so that the obtained rare earth protein fiber has excellent mechanical properties of high strength and high toughness. The rare earth protein fiber can be used in the fields of three-dimensional cell culture, tissue engineering and the like. The invention also provides a preparation method of the rare earth protein fiber, and the preparation method is simple and easy to industrialize.
Description
Technical Field
The invention belongs to the technical field of bioengineering materials, and particularly relates to a rare earth protein fiber and a preparation method thereof.
Background
Protein fibers produced from many protein sources, including plants, insects, and animals, are of widespread interest. Although natural protein fibers were used long ago, the development of artificial protein fibers began well in the 50's of the 20 th century. Protein fibers have become very important in the development of lightweight and high-strength materials because they are not only lightweight, but also biodegradable, have good moisture and temperature regulation, are resistant to high temperatures, are elastic, and have specific mechanical properties.
The nano composite material prepared from the artificial protein fiber and other polymers has great application potential in the aspects of textile, health and medical treatment, energy and engineering application, and can be used for preparing novel light high-strength functional materials. Despite some advances, artificial protein fibers still do not compare to natural spider silk in terms of toughness and rigidity, which limits their further applications. In addition, it is still rarely reported how to improve the mechanical properties of the artificial protein fiber by using the interaction between supramolecules. Therefore, there is a need to develop a technology capable of controlling the mechanical properties of the bioprotein fiber so as to better apply the prepared fiber to the high-tech field.
Disclosure of Invention
The invention provides a rare earth protein fiber and a preparation method thereof.
The invention provides a rare earth protein fiber, which is prepared by drawing super positive charge protein and anionic surfactant;
the anionic surfactant has a catechol building block.
Preferably, the rare earth protein fiber is made of super positive charge protein, anionic surfactant and metal ions;
the metal ion is Fe3+And/or Tb3+。
Preferably, the super-positively charged protein is expressed according to the following steps:
transforming the plasmid into an expression strain pichia pastoris or escherichia coli expression strain, and picking the monoclonal bacteria on the plate to fall into an LB culture medium for overnight activation; adding overnight activated bacterial liquid into TB culture medium, adding amino acid when od value is 1, cooling to 30 deg.C, leaking and expressing, collecting thallus overnight, resuspending with lysis buffer, adding protease inhibitor, DNase, lysozyme, MgCl, etc2Crushing thallus with a high pressure homogenizer, centrifuging at a speed of more than 10000rpm to obtain protein supernatant, and purifying by HPLC to obtain super positive charge protein.
Preferably, the charge number of the super positive charge protein is 72-108.
Preferably, the anionic surfactant is 6- (3- (3, 4-o-diphenol) propionamide) hexyl sodium sulfate.
Preferably, the molar ratio of the super positive charge protein to the anionic surfactant is (0.1-2): 4.
preferably, the molar ratio of the anionic surfactant to the metal ion is 1: (3-5).
The invention provides a preparation method of rare earth protein fiber, which comprises the following steps:
A) respectively dissolving super positive charge protein and an anionic surfactant in ultrapure water to obtain a super positive charge protein solution and an anionic surfactant solution;
the anionic surfactant has a catechol structural unit;
B) dropwise adding the anionic surfactant solution into the super positive charge protein solution, and uniformly mixing to obtain a mixed solution;
C) centrifuging the adhesive solution, and freeze-drying for 0.5-1 hour to obtain a rare earth protein mixture;
D) drawing the rare earth protein mixture to obtain rare earth protein fibers;
preferably, the speed of the centrifugation is 15000-18000 rpm;
the centrifugation time is 5-20 min.
Preferably, the water content of the rare earth protein mixture is 10-50%.
The invention provides a rare earth protein fiber, which is prepared by drawing super positive charge protein and anionic surfactant; the anionic surfactant has a catechol building block. The super positive charge protein and the anionic surfactant form a protein-surfactant compound under the electrostatic action, and different metal ions are doped to be chelated with the protein-surfactant compound for coordination crosslinking, so that the obtained rare earth protein fiber has excellent mechanical properties of high strength and high toughness. The rare earth protein fiber can be used in the fields of three-dimensional cell culture, tissue engineering and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic representation of a protein fiber in example 1 of the present invention;
FIG. 2 is a microscope photograph of protein fibers in example 1 of the present invention;
FIG. 3 is a polarized light microscope photograph of a protein fiber in example 1 of the present invention;
FIG. 4 is a graph showing the mechanical properties of the protein fiber in example 1 of the present invention;
FIG. 5 is a graph showing the mechanical properties of the protein fiber in example 2 of the present invention;
FIG. 6 is a graph showing mechanical properties of a rare earth protein fiber in example 3 of the present invention;
FIG. 7 is a schematic diagram of the action mechanism of the rare earth protein fiber in example 3 of the present invention, wherein the curve contacting with positive charge represents the protein sequence skeleton.
Detailed Description
The invention provides a rare earth protein fiber, which is prepared by drawing super positive charge protein and anionic surfactant;
the anionic surfactant has a catechol building block.
In the invention, the diameter of the rare earth protein fiber is preferably 15-30 μm.
In the present invention, the super-positively charged protein is preferably expressed according to the following steps:
transforming the gene vector of the supercharged protein K series into pichia pastoris (GS115/X-33/KM71) or escherichia coli expression strain (BL21/BL21DE3/BLRDE3), selecting a monoclonal colony, and carrying out overnight culture by using LB culture solution; adding the overnight activated expression seed liquid into a TB culture medium, adding an inducer isopropyl-beta-D-thiogalactopyranoside when the bacterial liquid reaches an OD600 value of 0.8, and cooling to 30 ℃ for overexpression. Inducing for 12 hours, collecting thalli, resuspending with lysis buffer solution, adding protease inhibitor, DNA enzyme and lysozyme, crushing thalli with a high-pressure crusher, centrifuging at a rotating speed of more than 10000rpm, collecting supernate, and purifying by HPLC to obtain the super positive charge protein.
In the invention, the charge number of the super positive charge protein is preferably 72-108; the super positive charge protein is a non-folding super positive charge protein.
In the present invention, the anionic surfactant has a catechol structural unit, preferably sodium 6- (3- (3, 4-o-diphenol) propionamide) hexyl sulfate.
In the present invention, the rare earth protein fiber preferably further comprises metal ions, and the metal ions are preferably Fe3+And/or Tb3+。
In the invention, the molar ratio of the super-positive charge protein (calculated by lysine) to the anionic surfactant is preferably (0.1-2): 4, more preferably (1 to 1.5): 4; the molar ratio of the anionic surfactant to the metal ion is preferably 1: (3-5), more preferably 1: (3-4).
In the invention, the high-strength rare earth protein fiber is prepared from the aqueous solutions of the three components, and the water content in the finally obtained high-strength rare earth protein adhesive is preferably 10-50%, and more preferably 20-40%.
The invention can prepare two types of protein fibers with different internal structures, namely high-strength high-toughness protein fibers, wherein metal particles are not added, the protein fibers prepared by drawing the super positive charge protein and the anionic surfactant are high-toughness fibers, and the protein fibers added with the metal particles are high-strength fibers.
The invention also provides a preparation method of the rare earth protein fiber, which comprises the following steps:
A) respectively dissolving super positive charge protein and an anionic surfactant in ultrapure water to obtain a super positive charge protein solution and an anionic surfactant solution;
the anionic surfactant has a catechol structural unit;
B) dropwise adding the anionic surfactant solution into the super positive charge protein solution, and uniformly mixing to obtain a mixed solution;
C) centrifuging the adhesive solution, and freeze-drying for 0.5-1 hour to obtain a rare earth protein mixture;
D) drawing the rare earth protein mixture to obtain rare earth protein fibers;
in the present invention, the kind and preparation method of the super positive charge protein are the same as those of the super positive charge protein described above, and are not described herein again.
In the present invention, the anionic surfactant is preferably 6- (3- (3, 4-o-diphenol) propionamide) hexyl sodium sulfate, 3, 4-dihydroxyphenyl propionic acid is used as a starting material, and the anionic surfactant is purified by multi-step protection, deprotection, amination reaction, nucleophilic substitution reaction and column chromatography. The synthetic process route is well known to those skilled in the art and will not be described herein.
In the invention, the molar concentration of the super positive charge protein solution is preferably 200-300 mu mol/L, and more preferably 220-250 mu mol/L; the preferable molar concentration of the anionic surfactant solution is 10-20 mu mol/L.
In the invention, the anionic surfactant solution is dripped into the super positive charge protein solution, and after the mixture is uniformly mixed, ferric salt and/or terbium salt are preferably added to obtain a mixed solution.
The iron salt is preferably FeCl3(ii) a The terbium salt is preferably TbCl3。
In the present invention, the amounts of the super positive charge protein, the anionic surfactant and the metal ion are the same as those of the super positive charge protein, the anionic surfactant and the metal ion, and thus, the detailed description thereof is omitted.
After the mixed solution is obtained, the mixed solution is subjected to high-speed centrifugation and then is directly freeze-dried to obtain the rare earth protein mixture.
In the invention, the water content of the rare earth protein mixture is preferably 10-50%, more preferably 20-40%, and most preferably 30%.
In the invention, the speed of centrifugation is preferably 15000-18000 rpm, more preferably 16000-17000 rpm, and most preferably 16800 rpm; the time for centrifugation is preferably 5-20 min, and more preferably 10-15 min.
The freeze-drying time is preferably 0.5-1 hour.
After the rare earth protein mixture is obtained, the invention manually performs wire drawing on the rare earth protein mixture to obtain the rare earth protein fiber. By controlling the drawing rate, the diameter of the fiber can be regulated.
The method for drawing the wire is not particularly limited, and manual wire drawing or electric motor wire drawing can be adopted.
The invention provides a rare earth protein fiber, which is prepared by drawing super positive charge protein and anionic surfactant; the anionic surfactant has a catechol building block. The super positive charge protein and the anionic surfactant form a protein-surfactant compound under the electrostatic action, and different metal ions are doped to be chelated with the protein-surfactant compound for coordination crosslinking, so that the obtained rare earth protein fiber has excellent mechanical properties of high strength and high toughness. The rare earth protein fiber can be used in the fields of three-dimensional cell culture, tissue engineering and the like.
In order to further illustrate the present invention, the following detailed description of a rare earth protein fiber and a method for preparing the same is provided in connection with examples, which should not be construed as limiting the scope of the present invention.
Example 1
Transforming the supercharged protein K series gene vector into an escherichia coli expression strain BLRDE3, selecting a monoclonal colony, and carrying out overnight culture by using LB culture solution; adding the overnight activated expression seed liquid into a TB culture medium, adding an inducer isopropyl-beta-D-thiogalactopyranoside when the bacterial liquid reaches an OD600 value of 0.8, and cooling to 30 ℃ for overexpression. Inducing for 12 hours, collecting thalli, resuspending with lysis buffer solution, adding protease inhibitor, DNA enzyme and lysozyme, crushing thalli with a high-pressure crusher, centrifuging at a rotating speed of more than 10000rpm, collecting supernate, and purifying by HPLC to obtain the super positive charge protein.
Weighing the super-charge K-series protein and the surfactant according to the molar ratio of 1:1, respectively dissolving the super-charge K-series protein and the surfactant in ultrapure water, and respectively preparing a super-charge protein aqueous solution with the concentration of about 220 mu mol/L and a surfactant aqueous solution with the concentration of 10 mu mol/L. The protein-surfactant complex can be obtained by mixing the two, shaking for 10 minutes and directly freeze-drying for 0.5 hour.
The protein-surfactant complex was manually drawn to obtain a protein fiber having a diameter of 20 μm and a water content of 45%.
As can be seen from fig. 4, at high water content, the produced protein fiber has ultra-high ductility and resilience.
Example 2
Protein fibers were prepared according to the preparation method of example 1 except that the lyophilization time was 1 hour, and the obtained protein fibers had a water content of 10% and a diameter of 20 μm.
As can be seen from FIG. 5, at low water content, the prepared protein fiber has poor ductility, but the corresponding mechanical properties including Young's modulus and tensile strength are enhanced to some extent.
Example 3
Transforming the plasmid into an escherichia coli expression strain BL21DE3, and picking monoclonal bacteria on the plate to fall into an LB culture medium for overnight activation; adding overnight activated bacterial liquid into TB culture medium, adding amino acid when od value is 1, cooling to 30 deg.C, leaking and expressing, collecting thallus overnight, resuspending with lysis buffer, adding protease inhibitor, DNase, lysozyme, MgCl, etc2Crushing thallus with a high pressure homogenizer, centrifuging at a speed of more than 10000rpm to obtain protein supernatant, and purifying by HPLC to obtain super positive charge protein (K) with a charge number of 108.
Weighing the super-charge K-series protein and the surfactant according to the molar ratio of 1:1, respectively dissolving the super-charge K-series protein and the surfactant in ultrapure water, respectively preparing a super-charge protein aqueous solution with the concentration of about 220 mu mol/L and a surfactant aqueous solution with the concentration of 10 mu mol/L, dropwise adding the surfactant aqueous solution into the super-charge K-series protein aqueous solution, uniformly mixing, and adding FeCl3Thus obtaining a corresponding mixture of aqueous solution, Fe3+The molar ratio of the total moles of (a) to the surfactant is 1: 3. Shaking for 10 min, and directly freeze-drying for 0.5 h to obtain the protein-surfactant complex.
The protein-surfactant complex is manually drawn to obtain protein fibers with the diameter of 20 mu m and the water content of 8 percent.
As can be seen from FIG. 6, after the metal coordination, the prepared protein fiber is characterized by brittleness, poor ductility, and significantly enhanced mechanical properties including Young's modulus and tensile strength.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. A protein fiber is prepared by drawing super positive charge protein and anionic surfactant;
the preparation method comprises the following specific steps:
A) respectively dissolving super positive charge protein and an anionic surfactant in ultrapure water to obtain a super positive charge protein solution and an anionic surfactant solution;
the anionic surfactant is 6- (3- (3, 4-o-diphenol) propionamide) hexyl sodium sulfate; the molar ratio of the super positive charge protein to the anionic surfactant is (0.1-2): 4;
B) dropwise adding the anionic surfactant solution into the super positive charge protein solution, and uniformly mixing to obtain a mixed solution;
C) centrifuging the mixed solution, and freeze-drying for 0.5-1 hour to obtain a protein-surfactant compound;
D) drawing the protein-surfactant compound to obtain protein fibers;
the super positive charge protein is expressed according to the following steps:
transforming the plasmid into a pichia pastoris or escherichia coli expression strain, and picking the monoclonal bacteria on the plate to fall into an LB culture medium for overnight activation; adding overnight activated bacterial liquid into TB culture medium, adding amino acid when od value is 1, cooling to 30 deg.C, leaking and expressing, collecting thallus overnight, resuspending with lysis buffer, adding protease inhibitor, DNase, lysozyme, MgCl, etc2Crushing thallus by using a high-pressure homogenizer, centrifuging at a speed of more than 10000rpm to obtain protein supernatant, and purifying by using HPLC to obtain super positive charge protein;
the charge number of the super positive charge protein is 72-108.
2. The protein fiber according to claim 1, wherein the protein fiber is made of a super-positively charged protein, an anionic surfactant, and metal ions;
the step B) is as follows: dropwise adding the anionic surfactant solution into the super positive charge protein solution, uniformly mixing, and adding ferric salt and/or terbium salt to obtain a mixed solution;
the metal ion is Fe3+And/or Tb3+。
3. The protein fiber according to claim 2, wherein the molar ratio of anionic surfactant to metal ion is 1: (3-5).
4. The protein fiber according to claim 1, wherein the centrifugation rate is 15000 to 18000 rpm;
the centrifugation time is 5-20 min.
5. The protein fiber according to claim 1, wherein the protein-surfactant complex has a water content of 10 to 50%.
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| CN101978040A (en) * | 2008-01-24 | 2011-02-16 | 犹他卅大学研究基金会 | Adhesive complex coacervates and methods of making and using thereof |
| CN106636016A (en) * | 2017-02-24 | 2017-05-10 | 天津大学 | Method for assisting self-assembly of virus-like particles by introducing positive and negative charges and application |
| CN109913977A (en) * | 2019-03-15 | 2019-06-21 | 中国科学院长春应用化学研究所 | A kind of nucleic acid gel fiber and preparation method thereof |
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| JPWO2017110922A1 (en) * | 2015-12-25 | 2018-10-18 | Ｓｐｉｂｅｒ株式会社 | Method for producing polymer aggregate |
| KR102010051B1 (en) * | 2017-09-20 | 2019-08-12 | 주식회사 포스코 | Coacervate formed from catechol conjugated anionic polymer, and adhesive comprising thereof and method for preparing the same |
| CN109912720B (en) * | 2019-03-14 | 2021-12-07 | 天津大学 | Design and synthesis method and spinning of spider silk protein |
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| CN101978040A (en) * | 2008-01-24 | 2011-02-16 | 犹他卅大学研究基金会 | Adhesive complex coacervates and methods of making and using thereof |
| CN106636016A (en) * | 2017-02-24 | 2017-05-10 | 天津大学 | Method for assisting self-assembly of virus-like particles by introducing positive and negative charges and application |
| CN109913977A (en) * | 2019-03-15 | 2019-06-21 | 中国科学院长春应用化学研究所 | A kind of nucleic acid gel fiber and preparation method thereof |
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