CN110551194B - Recombinant spider ootheca silk protein compound and artificial ootheca silk generated by same - Google Patents

Recombinant spider ootheca silk protein compound and artificial ootheca silk generated by same Download PDF

Info

Publication number
CN110551194B
CN110551194B CN201910884960.1A CN201910884960A CN110551194B CN 110551194 B CN110551194 B CN 110551194B CN 201910884960 A CN201910884960 A CN 201910884960A CN 110551194 B CN110551194 B CN 110551194B
Authority
CN
China
Prior art keywords
ser
ala
leu
gly
thr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910884960.1A
Other languages
Chinese (zh)
Other versions
CN110551194A (en
Inventor
林志
白向丽
范天天
袁文肃
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tianjin University
Original Assignee
Tianjin University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin University filed Critical Tianjin University
Priority to CN201910884960.1A priority Critical patent/CN110551194B/en
Publication of CN110551194A publication Critical patent/CN110551194A/en
Application granted granted Critical
Publication of CN110551194B publication Critical patent/CN110551194B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F8/00—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof
    • D01F8/02—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof from cellulose, cellulose derivatives, or proteins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Insects & Arthropods (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Textile Engineering (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to a recombinant spider ootheca silk protein complexAnd the artificial coleus; the compound is composed of a molecular framework NTD- (RP 1) m ‑(RP2) n CTD and the molecular framework is (RP) q Composition is carried out; constructing two types of recombinant spider silk protein expression plasmids, and respectively performing over-expression on recombinant spider silk proteins in escherichia coli, wherein the molar ratio of a recombinant egg sheath silk protein monomer I to a monomer II is 3-25; freeze drying the above compound, and dissolving with hexafluoroisopropanol to form spidroin protein solution; then spinning by wet spinning in a coagulation bath containing zinc chloride and ferric chloride. The tensile strength of the artificial egg sheath filaments manufactured by the recombinant egg sheath filament protein compound is averagely improved by about 35 percent and exceeds the strength of natural egg sheath filaments of the same type.

Description

Recombinant spider ootheca silk protein compound and artificial ootheca silk generated by same
Technical Field
The invention relates to a recombinant spider ootheca silk protein compound and an artificial ootheca silk generated by the same; in particular, a recombinant spider ootheca silk protein complex and a method for manufacturing high-strength artificial ootheca silk using the same.
Background
Spider silks have excellent mechanical properties and a wide potential application prospect, and can be applied to the fields of textile industry, optics, environmental engineering, biomedicine, military industry and the like, such as microsurgical sutures, tissue engineering scaffolds, drug delivery carriers, hemostatic bandages, bulletproof materials and the like, wherein the materials have specific physical properties, such as certain strength, toughness, elasticity, biodegradability and biocompatibility, so spider silks meeting the conditions are focused (Vollrath, F. & Knight, d.p., nature,410, 541, 2001). However, spiders have the characteristic of cannibalism and cannot be raised on a large scale. Therefore, the production mechanism of spider silk has been intensively studied, attempting to produce artificial spider silk proteins by techniques such as genetic engineering, protein engineering or synthetic biology, and to artificially spin artificial spider silk in vitro, simulating the spinners and filamentation conditions of spiders (Lazaris, a.et al, science,2002,295, 472, teule, f.et al, nat. Protoc.,2009,4, scheller, j.et al, nat. Biotechnol.,2001, 19.
Spider silk proteins that make up spider silks are composed of a central multiple repeating domain and two terminal non-repeating domains, with high Molecular Weights (MW) ranging from-200 kDa to-1380 kDa (Chen g.et al.plos One,2012, 7; furthermore, spidroins of the same type are typically composed of multiple spidroin proteins (Hu x.et al.biochemistry,2006,45, 3506, huang w.et al.sci. Rep.,2017, 7. This means that the size and species make-up of spider silk proteins are key determinants of the excellent mechanical properties of spider silks. Researchers are currently primarily producing single recombinant spidroin proteins by expression systems such as e.coli, yeast, plant cells, insect cells and mammalian cells (Hauptmann v.et al. Transgenic res, 2013, 22. However, the expression of a foreign protein having a high molecular weight (e.g., more than 200 kD) in E.coli results in a serious decrease in the expression level. In order to solve the problem of in vitro expression of large molecular weight spidroin proteins, researchers have attempted to obtain a mixture of 2 to 10 domains of polymeric recombinant silk proteins using plant expression systems, but have not been able to obtain homogeneous high molecular weight recombinant silk proteins (Hauptmann v. Et al. Transgenic res.,2013, 369.
PCT patent document No. WO2007/078239 discloses a method for expressing high molecular weight recombinant dragline silk protein (about 285 kDa) by using a modified Escherichia coli BL21 (DE 3) strain, and artificially spinning artificial protein silk with good mechanical properties by using the single recombinant protein. But the breaking strength is still inferior to that of the natural traction wire.
Another approach is to covalently link different spidroin proteins together by disulfide bonds to increase the molecular weight. Non-specific linking can only be used to produce non-uniform polymers, and the high molecular weight polymer content is too low, so that the mechanical properties of rayon fibers are only slightly improved (Grip s.et al. Protein sci.,2010, 18. But linked specifically by the terminal domain, a homogeneous high molecular weight recombinant silk protein (about 378 kDa) can be produced from which synthetic filaments can be spun that are comparable in tensile strength to the corresponding native spider silk (Lin z.et al.adv. Mater, 2013, 1216. Recently, it has been possible to produce artificial dragline yarns with high performance using an ultra-high molecular weight recombinant dragline silk protein (about 556 kDa) formed by intein cleavage ligation, which has been close to the strength of natural dragline yarns in tensile strength and elongation (Bowen c.h.et al. Biomacromolecules,2018, 19. In summary, there are great difficulties in the current mass production of rayon spun from high molecular weight spidroin proteins; furthermore, the production of artificial spider silks relies mainly on a single recombinant silk protein, which may also be one of the main reasons why one cannot prepare artificial silks with excellent mechanical properties under mild or weakly toxic conditions.
According to the present research it has been shown that, isolated from spider coleus ovales the spidroin protein mainly comprises TuSp (Tubuliform spidrin) (Zhao a.et al.biochemistry,2006, 45; huang W.et al Biochimie,2006, 88. In the coleus of Latrodectus hesperus, the black widow spider, two additional different silk proteins are included: ECP-1 (Egg case pProtein-1) and ECP-2 (Hu x. Et al. Biochemistry,2006, 45. In the golden cylinder spider Nephila antipodaiana, ootheca silks have been identified to contain at least two silk proteins TuSp1 and TuSp2 (Huang w.et al. Sci. Rep.,2017, 13354. TuSp1 is the core protein of the silk of the egg sheath, and is also the main component, and the whole length molecular weight is about 360kDa, but the complete sequence is not known. Silk amino acid sequence and higher structure studies indicate that TuSp1 contains multiple conserved repeat domains (TuSp 1 recurring domain, tuSp 1-RP), as well as one amino terminal (TuSp 1N-terminal domain, tuSp 1-NTD) and one carboxy terminal non-repeat domain (TuSp 1C-terminal domain, tuSp 1-CTD) (Lin z. Et al. Proc. Natl. Acad. Sci. Usa,2009, 106. TuSp1-RP can be divided into two types, i.e. a type I repeat domain (TuSp 1-RP 1) and a type II repeat domain (TuSp 1-RP 2); the repeat sequence is high in alanine and serine (about 40-50% total), unlike the pull and minor ampullate gland filaments, which are rich in alanine and glycine. Another kind of tunicasin TuSp2 is tunicasinThe non-main component (2) of (2), which similarly contains a plurality of repeating domains (TuSp 2-RP), does not have a typical carboxyl-terminal non-repeating domain of silk protein, and the amino-terminal sequence thereof is not known. The structure is rich in alanine, serine, asparagine and leucine (total about 45%). Therefore, in order to obtain the artificial ootheca silk with excellent mechanical properties, the invention designs a compound containing two different types of recombinant ootheca silk proteins based on the current research on the molecular structure of the ootheca silk protein of the golden cylinder spider, and produces the artificial ootheca silk with more excellent mechanical properties than the existing artificial ootheca silk by artificial spinning, and the artificial ootheca silk also exceeds the natural ootheca silk in tensile strength.
Disclosure of Invention
The first object of the present invention is to provide a recombinant silk protein complex for producing an artificial spider ootheca silk. The second object of the present invention is to obtain a novel artificial coleus thread having a strength significantly improved as compared with a single recombinant protein by forming a thread in a coagulation solution having low toxicity using the recombinant silk protein complex as a raw material.
The technical scheme of the invention is as follows:
a recombinant spider coleoptilin complex; consists of a recombinant egg sheath silk protein monomer I and a monomer II, wherein the molecular framework of the recombinant egg sheath silk protein monomer I is NTD- (RP 1) m -(RP2) n -CTD, wherein the integer m represents the repetition number of TuSp1-RP1 in TuSp1 type i repeating domain, n represents the repetition number of TuSp1-RP2 in type ii repeating domain, and m and n both range from 1 to 40; the molecular framework of the recombinant egg sheath silk protein monomer II is (RP) q Wherein, the integer q represents the repetition times of TuSp2-RP in the TuSp2 repeat structural domain, and the value range is 1-40.
The recombinant spider ootheca silk protein complex; the single TuSp1-RP1 amino acid sequence is shown as SEQ ID NO. 1, and the single TuSp1-RP2 amino acid sequence is shown as SEQ ID NO. 2; the recombinant ovalbumin monomer I also comprises a TuSp1 amino-terminal non-repetitive domain TuSp1-NTD, and the amino acid sequence is shown in SEQ ID NO. 3; and a TuSp1 carboxy-terminal non-repetitive domain TuSp1-CTD, the amino acid sequence of which is shown in SEQ ID NO. 4.
The recombinant spider ootheca silk protein complex; the single TuSp2-RP amino acid sequence is shown in SEQ ID NO. 5.
The recombinant spider ootheca silk protein complex; the molecular framework is NTD- (RP 1) m -(RP2) n -the sum of m and n in the CTD is less than or equal to 40.
The recombinant spider ootheca silk protein complex; each domain of which has a structure with a serine content of 10% or more.
The method for artificially synthesizing the spider ootheca silks by using the recombinant spider ootheca silk protein compound; firstly, constructing two types of recombinant spider silk protein expression plasmids, respectively overexpressing recombinant spider silk proteins in escherichia coli, mixing the two recombinant spider silk proteins after expression and purification to form a compound, wherein the molar ratio of a recombinant lecithin protein monomer I to a monomer II is 3-25; the above complex was freeze-dried and dissolved in hexafluoroisopropanol, and then wet-spun in a coagulation bath containing zinc chloride and ferric chloride.
The domain sequences involved in the present invention are illustrated in Table 1:
TABLE 1
Figure BDA0002207027940000031
Compared with the existing single type of artificial ootheca silk, the invention has the beneficial effect that the mechanical property of the artificial ootheca silk prepared by utilizing the recombinant ootheca silk protein compound is greatly improved. The molar ratio of the recombinant egg sheath silk protein monomer I to the monomer II is 6:1, the tensile strength is averagely improved by about 35 percent and exceeds the strength of the natural ootheca filaments of the same type. In addition, the inorganic zinc/iron salt solution is used as a coagulating bath and the alcohol solution is used as a post-treatment reagent in the process of manufacturing the artificial ootheca silk, so that the artificial ootheca silk is more environment-friendly compared with high-toxicity organic reagents such as isopropanol, methanol and the like used in the previous research, and further possibility is provided for industrial mass production of the artificial ootheca silk.
Drawings
FIG. 1 shows a schematic representation of the molecular architecture of the recombinant spidroin TuSp1-N2RPC and TuSp 2-RP.
FIG. 2 shows an expression system for expressing recombinant monomer of a silk fibroin and a recombinant module of silk fibroin on an expression vector.
FIG. 3 is a SDS-PAGE analysis of recombinant spidroin TuSp1-N2RPC and TuSp2-RP purified samples, wherein M is a protein molecular weight standard; lane 1 is a TuSp1-N2RPC purified sample; lane 2 is a TuSp2-RP purified sample.
FIG. 4 is a schematic view of a spinning apparatus.
FIG. 5 shows images of an artificial ootheca silk formed from TuSp1-N2RPC TuSp2-RP composite (molar ratio 6:1) using a conventional optical microscope (a) and a polarizing microscope (b) at a scale bar of 50 μm.
Fig. 6 is a graph comparing stress curves of artificial ootheca filaments. i, a control group TuSp1-N2RPC; ii to v, tuSp1-N2RPC: tuSp2-RP, molar ratios 3:1 (ii), 6:1 (iii), 12.
FIG. 7 is a graph showing the comparison among the tensile strength (a), breaking energy (b) and Young's modulus (c) of an artificial coleus filament. i, a control group TuSp1-N2RPC; ii-v, tuSp1-N2RPC TuSp2-RP, molar ratios 3:1 (ii), 6:1 (iii), 12.
Detailed Description
The following describes in detail embodiments of the present invention in examples in which the compound used to produce the synthetic spider silk comprises two recombinant proteins, tuSp1-N2RPC (recombinant silk fibroin monomer i) and TuSp2-RP (recombinant silk fibroin monomer ii), whose molecular architectures are NTD- (RP 1) 1 -(RP2) 1 CTD and (RP) 1 (as shown in fig. 1). Wherein TuSp1-N2RPC is a recombinant spidroin protein from TuSp1, consisting of four different TuSp1 domains, one TuSp1-RP1 (corresponding to SEQ ID NO: 1), one TuSp1-RP2 (corresponding to SEQ ID NO: 2), one TuSp1-NTD (corresponding to SEQ ID NO: 3) and one TuSp1-CTD (corresponding to SEQ ID NO: 4), respectively. The serine content of the above four domains was 23%, 21% and 20%, respectively. TuSp2Recombinant spidroin protein with TuSp2 as RP, comprising only one TuSp2-RP (corresponding to SEQ ID NO: 5), with a serine content of 11%. The specific embodiment of the preparation of artificial spider ootheca silk in this example is as follows:
example 1: constructing Escherichia coli expression plasmids of recombinant spidroin TuSp1-N2RPC and TuSp 2-RP.
1.1 construction of recombinant ovalbumin expression plasmid pTuSp1-N2 RPC.
And (3) obtaining a TuSp1-N2RPC target gene. Based on previous studies on the molecular structure and gene sequence of the golden cylinder spider ootheca silk protein TuSp1 (Lin z. Et al. Proc. Natl. Acad. Sci. Usa,2009, 106). Finally, the constructed recombinant plasmid is confirmed by restriction enzyme digestion and base sequence analysis.
1.2 construction of recombinant ovalbumin expression plasmid pTuSp 2-RP.
Obtaining TuSp2-RP target gene. Based on the previous molecular structure study on TuSp2 of spidermus spiders of golden cylinder spider (Huang w. Sci. Rep.2017, 7. And finally, confirming the constructed recombinant plasmid through restriction enzyme digestion and base sequence analysis.
The strains, plasmid vectors, antibiotics and culture media involved in this example were: coli DH 5. Alpha., expression plasmid pET32a + (without Trx and S tags), ampicillin, LB medium (10 g/L tryptone, 5g/L yeast powder and 10g/L sodium chloride). All nucleic acid manipulation steps in this example were performed according to standard methods (Green M.R.et. Al., molecular cloning: a Laboratory Manual,4th Ed., cold Spring Harbor Laboratory Press, 2012).
Example 2: expression and purification of recombinant spider silk proteins.
The two recombinant spider silk proteins in the complex are expressed in an escherichia coli expression system respectively and purified by a nickel column affinity chromatography method.
2.1 Expression and purification of TuSp1-N2RPC recombinant spider silk protein
(1) And (4) preparing seed liquid. The recombinant plasmid containing pTuSp1-N2RPC was transformed into E.coli BL21 (DE 3) pLysS to form an expression strain. The above monoclonal strain was inoculated into TB liquid medium (12 g/L tryptone, 24g/L yeast powder, 4mL/L glycerol and 50mM phosphate buffer) having a pH of 6.8, added with 100. Mu.g/mL ampicillin and 34. Mu.g/mL chloramphenicol, and shake-cultured at 37 ℃ and 220rpm for 10 to 12 hours.
(2) And (4) inducing expression. The prepared seed solution was transferred to a fresh TB liquid medium to a pH of 6.8, added with 100. Mu.g/ml ampicillin and 34. Mu.g/ml chloramphenicol, shake-cultured at 37 ℃ and 220rpm to OD 600 1.2 to 2.8, adding isopropyl-beta-thiogalactopyranoside (IPTG) with the final concentration of 0.1mM, carrying out induced expression for 12 to 18 hours at the temperature of between 16 and 25 ℃, and collecting thalli.
(3) And (4) protein purification. And (3) resuspending the collected TuSp1-N2RPC thalli by using sterile water, and ultrasonically crushing the thalli by using an ultrasonic crusher in ice bath at the power of 270W for 20-45 minutes. And centrifuging to collect the ultrasonic cracking supernatant and the precipitate, denaturing the precipitate with 8M urea to be uniform and transparent, and centrifuging again to collect the denatured supernatant. The denatured supernatant contained TuSp1-N2RPC, which was purified, collected and dialyzed using a nickel column. The analysis result of the obtained recombinant silk protein TuSp1-N2RPC is shown in lane 1 of FIG. 3, and the purity is about 90%.
2.2 Expression and purification of TuSp2-RP recombinant spider silk proteins
(1) And (4) preparing seed liquid. The recombinant plasmid containing pTuSp2-RP was transformed into E.coli BL21 (DE 3) pLysS to form an expression strain. The above monoclonal strain was inoculated into LB medium (10 g/L tryptone, 5g/L yeast powder and 10g/L sodium chloride), added with 100. Mu.g/ml ampicillin and 34. Mu.g/ml chloramphenicol, and shake-cultured at 37 ℃ and 220rpm for 10-12 hours.
(2) And (3) inducing expression. Transferring the prepared seed liquid into a fresh LB culture medium, and addingAdding 100. Mu.g/ml ampicillin and 34. Mu.g/ml chloramphenicol, shaking-culturing at 37 deg.C and 180rpm to OD 600 0.5-1.0, adding IPTG with the final concentration of 0.1mM to induce expression for 12-18 hours at 16-25 ℃, and collecting thalli.
(3) And (4) protein purification. After the collected TuSp2-RP bacterial cells were resuspended in Tris-containing Lysis Buffer, the cells were disrupted by a low-temperature ultrahigh-pressure continuous flow cell disrupter, and the supernatant was collected by centrifugation, purified, collected and dialyzed using a nickel column. The analysis result of the obtained recombinant silk protein TuSp2-RP is shown in FIG. 3, lane 2, with a purity of about 90%.
Example 3 preparation and spinning process of artificial coleus spinning dope.
3.1 preparation of the spinning dope.
The purified recombinant spidroin TuSp1-N2RPC and TuSp2-RP are freeze-dried for 48 hours. Mixing TuSp1-N2RPC and TuSp2-RP in a certain proportion to form a composite. In the compound, the molar ratio of TuSp1-N2RPC to TuSp2-RP is minimum 3:1, max 25:1. and dissolving the compound with hexafluoroisopropanol at room temperature for 8 hours to form a uniform recombinant spider silk protein solution with the mass percentage of 10-15 percent, namely the spinning solution. The spinning solution of the reference group only contains single recombinant spidroin protein TuSp1-N2RPC with the mass percentage of 10-15%.
3.2 preparation of the nascent artificial spider ootheca silk fiber.
First, an aqueous solution containing 100mM zinc chloride and 2mM ferric chloride was prepared as a coagulation bath for synthesizing spiders' ootheca silks at a temperature of 25 ℃. Next, 500. Mu.l of the prepared spinning dope was transferred to a 1ml syringe, and a filament outlet having a diameter of about 127 μm was immersed in a coagulation bath in which a silk protein complex was formed into a filament. Finally, the nascent artificial spider ootheca silk formed in the coagulation bath is wound on a reel at a constant speed. A schematic of the spinning apparatus is shown in figure 4.
3.3 post-treatment of the artificial spider coleus ovalifolius.
The nascent artificial spider ootheca silk fiber has irregular internal molecular structure arrangement and poor mechanical property, and needs to be subjected to certain post-treatment to enhance the mechanical property. In the invention, the primary artificial spider ootheca silk fiber is immersed in 80-90% alcohol solution and is uniformly stretched 4-5 times at a constant speed of 3-4 mm/min, the primary artificial spider ootheca silk fiber is taken out and fixed, and the primary artificial spider ootheca silk fiber is dried in a room temperature environment to obtain the artificial spider ootheca silk fiber with the diameter of 5-8 mu m. A representative synthetic coleus filament fiber image of post-treatment is shown in figure 5; among them, the polarization micrographs show that rayon has significant birefringence, which indicates that silk protein molecules are well aligned in a specific direction. In this example, although the composites had different monomer ratios, no significant difference was seen in the micrographs of the silk fibers formed.
3.4 mechanical property test of the artificially synthesized spider ootheca silk.
The dried artificial ootheca silk fiber is fixed on a clamp, and the surface characteristics and the diameter of the artificial ootheca silk fiber are observed by using an optical microscope. We selected a sample (N = 3) with smooth surface and uniform diameter of 7-10 μm, and tested the mechanical properties on a universal tensile tester (E42.503, MTS) with a sensor with a rated load of 5N under the conditions of 40% relative humidity and room temperature: the gauge length is 20mm, and the stretching speed is 10mm/min. Fig. 6 shows two types of artificial coleus stress-strain curves.
All the measurements are shown in FIG. 7, and compared with the control group, i.e. the artificial coleus ovalbumus TuSp1-N2RPC containing only single recombinant silk protein, the mechanical properties (including tensile strength, breaking energy and Young modulus) of the artificial composite coleus TuSp1-N2RPC: tuSp2-RP (molar ratio 6:1) are improved; among them, the artificial coleus filaments having a tensile strength of 437. + -. 43MPa, which is improved by about 35% from that of the control group, are the highest tensile strength among the known artificial coleus filaments.
In summary, the artificial spider ootheca silk of the present invention is obtained by preparing a spinning solution from a complex of two recombinant spider ootheca silk proteins, and using a low-toxic and environmentally-friendly inorganic salt solution as a coagulation bath, and performing wet spinning. Compared with the artificial spider silk formed by single recombinant egg sheath silk protein, the mechanical property of the artificial spider silk is obviously improved. The design of the silk protein of the invention is based on the research on the structure and function of the natural silk protein of the egg sheath to select and combine the necessary functional modules. Since neither recombinant coleus proteins have a high molecular weight, they can be highly expressed in conventional E.coli expression systems and can be mixed in any ratio to form a rayon with specific physical properties before spinning. The technical features of the above-described embodiments should be considered as being within the scope of the present specification unless there is any contradiction. The above examples only express embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. In addition, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Sequence listing
SEQ ID NO:1
Ser Ala Ser Arg Ala Gly Ser Thr Ser Ser Ser Thr Thr Thr Thr Thr Ser Gly Ala Thr Ser Gln Ala Ala Ser Gln Ser Ala Ser Ser Ser Tyr Ser Ser Ala Phe Ala Gln Ala Ala Ser Ser Ala Leu Ala Thr Ser Ser Ala Ile Ser Arg Ala Phe Ala Ser Val Ser Ser Ala Ser Ala Ala Ser Ser Leu Ala Tyr Asn Ile Gly Leu Ser Ala Ala Arg Ser Leu Gly Ile Ala Ser Asp Thr Ala Leu Ala Gly Ala Leu Ala Gln Ala Val Gly Gly Val Gly Ala Gly Ala Ser Ala Ser Ala Tyr Ala Asn Ala Ile Ala Arg Ala Ala Gly Gln Phe Leu Ala Thr Gln Gly Val Leu Asn Ala Gly Asn Ala Ser Ala Leu Ala Gly Ser Phe Ala Arg Ala Leu Ser Ala Ser Ala Glu Ser Gln Ser Phe Ala Gln Ser Gln Ala Tyr Gln Gln Ala Ser Ala Phe Gln Gln Ala Ala Ala Gln Ser Ala Ala Gln
SEQ ID NO:2
Ser Ala Ser Arg Ala Gly Ser Thr Ser Ser Ser Thr Thr Thr Thr Thr Ser Gly Ala Thr Ser Gln Ala Ala Ser Gln Ser Ala Ser Ser Ser Tyr Ser Ser Ala Phe Ala Gln Ala Ala Ser Ser Ser Leu Ala Thr Ser Ser Ala Ile Ser Arg Ala Phe Ala Ser Val Ser Ser Ala Ser Ala Ala Ser Ser Leu Ala Tyr Asn Ile Gly Leu Ser Ala Ala Arg Ser Leu Gly Ile Ala Ser Asp Thr Ala Leu Ala Gly Ala Leu Ala Gln Ala Val Gly Gly Val Gly Ala Gly Ala Ser Ala Ser Ala Tyr Ala Asn Ala Ile Ala Arg Ala Ala Gly Gln Phe Leu Ala Thr Gln Gly Val Leu Asn Ala Val Asn Ala Ser Ser Leu Gly Ser Ala Leu Ala Asn Ala Leu Ser Asp Ser Ala Ala Asn Ser Ala Val Ser Gly Asn Tyr Leu Gly Val Ser Gln Asn Phe Gly Arg Ile Ala Pro Val Thr Gly Gly Thr Ala
SEQ ID NO:3
Gln Ala Ile Ser Val Ala Thr Ser Val Pro Ser Val Phe Ser Ser Pro Ser Leu Ala Ser Gly Phe Leu Gly Cys Leu Thr Thr Gly Ile Gly Gln Ser Pro Asp Phe Pro Phe Gln Glu Gln Gln Asp Leu Asp Asp Leu Ala Gln Val Ile Leu Ser Ala Val Thr Ser Asn Thr Asp Thr Ser Lys Ser Ala Arg Ala Gln Ala Leu Ser Thr Ala Leu Ala Ser Ser Leu Ala Asp Leu Leu Ile Ser Glu Ser Ser Gly Ser Ser Tyr Gln Thr Gln Ile Ser Ala Leu Thr Asn Ile Leu Ser Asp Cys Phe Val Thr Thr Thr Gly Ser Asn Asn Pro Ala Phe Val Ser Arg Val Gln Thr Leu Ile Ala Val Leu Ser Gln Ser Ser Ser Asn Ala Ile Ser Gly Ala Thr Gly Gly Ser Ala Phe Ala Gln Ser Gln Ala Phe Gln Gln Ser Ala Ser Gln Ser Ala Gly Leu
SEQ ID NO:4
Gly Ile Ser Val Gly Val Pro Gly Tyr Leu Arg Thr Pro Ser Ser Thr Ile Leu Ala Pro Ser Asn Ala Gln Ile Ile Ser Leu Gly Leu Gln Thr Thr Leu Ala Pro Val Leu Ser Ser Ser Gly Leu Ser Ser Ala Ser Ala Ser Ala Arg Val Ser Ser Leu Ala Gln Ser Leu Ala Ser Ala Leu Ser Thr Ser Arg Gly Thr Leu Ser Leu Ser Thr Phe Leu Asn Leu Leu Ser Ser Ile Ser Ser Glu Ile Arg Ala Ser Thr Ser Leu Asp Gly Thr Gln Ala Thr Val Glu Val Leu Leu Glu Ala Leu Ala Ala Leu Leu Gln Val Ile Asn Gly Ala Gln Ile Thr Asp Val Asn Val Ser Ser Val Pro Ser Val Asn Ala Ala Leu Val Ser Ala Leu Val Ala
SEQ ID NO:5
Asn Leu Ser Ile Gly Asp Thr Thr Ser Ile Ile Gln Leu Phe Lys Asn Phe Thr Gly Pro Pro Ser Val Ala Thr Phe Ile Ser Asn Phe His Ser Ile Val Gln Ser Ser Lys Thr Leu Leu Asn Leu Phe Asp Val Ala Glu Glu Asn Pro Leu Glu Phe Ala Lys Cys Met Tyr Glu Leu Val Leu Lys Ser Ala Asn Ser Leu Gly Val Leu Asn Pro His Leu Ile Ala Asn Asn Ile Tyr Gln Ser Val Val Ser Asn Leu Asp Ile Leu His Ser Ser Ala Met Val Asn Leu Tyr Ala Asn Ala Met Ala Gly Ser Leu Phe Leu Glu Gly Ile Leu Asn Ser Asp Asn Ala Ala Thr Leu Ala Lys Lys Cys Ala Asn Asp Met Glu Ala Phe Ala Lys Lys Met Val Glu Ile Gly
SEQ ID NO:6
Gln Ala Ile Ser Val Ala Thr Ser Val Pro Ser Val Phe Ser Ser Pro Ser Leu Ala Ser Gly Phe Leu Gly Cys Leu Thr Thr Gly Ile Gly Gln Ser Pro Asp Phe Pro Phe Gln Glu Gln Gln Asp Leu Asp Asp Leu Ala Gln Val Ile Leu Ser Ala Val Thr Ser Asn Thr Asp Thr Ser Lys Ser Ala Arg Ala Gln Ala Leu Ser Thr Ala Leu Ala Ser Ser Leu Ala Asp Leu Leu Ile Ser Glu Ser Ser Gly Ser Ser Tyr Gln Thr Gln Ile Ser Ala Leu Thr Asn Ile Leu Ser Asp Cys Phe Val Thr Thr Thr Gly Ser Asn Asn Pro Ala Phe Val Ser Arg Val Gln Thr Leu Ile Ala Val Leu Ser Gln Ser Ser Ser Asn Ala Ile Ser Gly Ala Thr Gly Gly Ser Ala Phe Ala Gln Ser Gln Ala Phe Gln Gln Ser Ala Ser Gln Ser Ala Gly Leu Ser Ala Ser Arg Ala Gly Ser Thr Ser Ser Ser Thr Thr Thr Thr Thr Ser Gly Ala Thr Ser Gln Ala Ala Ser Gln Ser Ala Ser Ser Ser Tyr Ser Ser Ala Phe Ala Gln Ala Ala Ser Ser Ala Leu Ala Thr Ser Ser Ala Ile Ser Arg Ala Phe Ala Ser Val Ser Ser Ala Ser Ala Ala Ser Ser Leu Ala Tyr Asn Ile Gly Leu Ser Ala Ala Arg Ser Leu Gly Ile Ala Ser Asp Thr Ala Leu Ala Gly Ala Leu Ala Gln Ala Val Gly Gly Val Gly Ala Gly Ala Ser Ala Ser Ala Tyr Ala Asn Ala Ile Ala Arg Ala Ala Gly Gln Phe Leu Ala Thr Gln Gly Val Leu Asn Ala Gly Asn Ala Ser Ala Leu Ala Gly Ser Phe Ala Arg Ala Leu Ser Ala Ser Ala Glu Ser Gln Ser Phe Ala Gln Ser Gln Ala Tyr Gln Gln Ala Ser Ala Phe Gln Gln Ala Ala Ala Gln Ser Ala Ala Gln Ser Ala Ser Arg Ala Gly Ser Thr Ser Ser Ser Thr Thr Thr Thr Thr Ser Gly Ala Thr Ser Gln Ala Ala Ser Gln Ser Ala Ser Ser Ser Tyr Ser Ser Ala Phe Ala Gln Ala Ala Ser Ser Ser Leu Ala Thr Ser Ser Ala Ile Ser Arg Ala Phe Ala Ser Val Ser Ser Ala Ser Ala Ala Ser Ser Leu Ala Tyr Asn Ile Gly Leu Ser Ala Ala Arg Ser Leu Gly Ile Ala Ser Asp Thr Ala Leu Ala Gly Ala Leu Ala Gln Ala Val Gly Gly Val Gly Ala Gly Ala Ser Ala Ser Ala Tyr Ala Asn Ala Ile Ala Arg Ala Ala Gly Gln Phe Leu Ala Thr Gln Gly Val Leu Asn Ala Val Asn Ala Ser Ser Leu Gly Ser Ala Leu Ala Asn Ala Leu Ser Asp Ser Ala Ala Asn Ser Ala Val Ser Gly Asn Tyr Leu Gly Val Ser Gln Asn Phe Gly Arg Ile Ala Pro Val Thr Gly Gly Thr Ala Gly Ile Ser Val Gly Val Pro Gly Tyr Leu Arg Thr Pro Ser Ser Thr Ile Leu Ala Pro Ser Asn Ala Gln Ile Ile Ser Leu Gly Leu Gln Thr Thr Leu Ala Pro Val Leu Ser Ser Ser Gly Leu Ser Ser Ala Ser Ala Ser Ala Arg Val Ser Ser Leu Ala Gln Ser Leu Ala Ser Ala Leu Ser Thr Ser Arg Gly Thr Leu Ser Leu Ser Thr Phe Leu Asn Leu Leu Ser Ser Ile Ser Ser Glu Ile Arg Ala Ser Thr Ser Leu Asp Gly Thr Gln Ala Thr Val Glu Val Leu Leu Glu Ala Leu Ala Ala Leu Leu Gln Val Ile Asn Gly Ala Gln Ile Thr Asp Val Asn Val Ser Ser Val Pro Ser Val Asn Ala Ala Leu Val Ser Ala Leu Val Ala。
Sequence listing
<110> Tianjin university
<120> recombinant spider ootheca silk protein complex and artificial ootheca silk produced therefrom
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 171
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Ser Ala Ser Arg Ala Gly Ser Thr Ser Ser Ser Thr Thr Thr Thr Thr
1 5 10 15
Ser Gly Ala Thr Ser Gln Ala Ala Ser Gln Ser Ala Ser Ser Ser Tyr
20 25 30
Ser Ser Ala Phe Ala Gln Ala Ala Ser Ser Ala Leu Ala Thr Ser Ser
35 40 45
Ala Ile Ser Arg Ala Phe Ala Ser Val Ser Ser Ala Ser Ala Ala Ser
50 55 60
Ser Leu Ala Tyr Asn Ile Gly Leu Ser Ala Ala Arg Ser Leu Gly Ile
65 70 75 80
Ala Ser Asp Thr Ala Leu Ala Gly Ala Leu Ala Gln Ala Val Gly Gly
85 90 95
Val Gly Ala Gly Ala Ser Ala Ser Ala Tyr Ala Asn Ala Ile Ala Arg
100 105 110
Ala Ala Gly Gln Phe Leu Ala Thr Gln Gly Val Leu Asn Ala Gly Asn
115 120 125
Ala Ser Ala Leu Ala Gly Ser Phe Ala Arg Ala Leu Ser Ala Ser Ala
130 135 140
Glu Ser Gln Ser Phe Ala Gln Ser Gln Ala Tyr Gln Gln Ala Ser Ala
145 150 155 160
Phe Gln Gln Ala Ala Ala Gln Ser Ala Ala Gln
165 170
<210> 2
<211> 171
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Ser Ala Ser Arg Ala Gly Ser Thr Ser Ser Ser Thr Thr Thr Thr Thr
1 5 10 15
Ser Gly Ala Thr Ser Gln Ala Ala Ser Gln Ser Ala Ser Ser Ser Tyr
20 25 30
Ser Ser Ala Phe Ala Gln Ala Ala Ser Ser Ser Leu Ala Thr Ser Ser
35 40 45
Ala Ile Ser Arg Ala Phe Ala Ser Val Ser Ser Ala Ser Ala Ala Ser
50 55 60
Ser Leu Ala Tyr Asn Ile Gly Leu Ser Ala Ala Arg Ser Leu Gly Ile
65 70 75 80
Ala Ser Asp Thr Ala Leu Ala Gly Ala Leu Ala Gln Ala Val Gly Gly
85 90 95
Val Gly Ala Gly Ala Ser Ala Ser Ala Tyr Ala Asn Ala Ile Ala Arg
100 105 110
Ala Ala Gly Gln Phe Leu Ala Thr Gln Gly Val Leu Asn Ala Val Asn
115 120 125
Ala Ser Ser Leu Gly Ser Ala Leu Ala Asn Ala Leu Ser Asp Ser Ala
130 135 140
Ala Asn Ser Ala Val Ser Gly Asn Tyr Leu Gly Val Ser Gln Asn Phe
145 150 155 160
Gly Arg Ile Ala Pro Val Thr Gly Gly Thr Ala
165 170
<210> 3
<211> 161
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Gln Ala Ile Ser Val Ala Thr Ser Val Pro Ser Val Phe Ser Ser Pro
1 5 10 15
Ser Leu Ala Ser Gly Phe Leu Gly Cys Leu Thr Thr Gly Ile Gly Gln
20 25 30
Ser Pro Asp Phe Pro Phe Gln Glu Gln Gln Asp Leu Asp Asp Leu Ala
35 40 45
Gln Val Ile Leu Ser Ala Val Thr Ser Asn Thr Asp Thr Ser Lys Ser
50 55 60
Ala Arg Ala Gln Ala Leu Ser Thr Ala Leu Ala Ser Ser Leu Ala Asp
65 70 75 80
Leu Leu Ile Ser Glu Ser Ser Gly Ser Ser Tyr Gln Thr Gln Ile Ser
85 90 95
Ala Leu Thr Asn Ile Leu Ser Asp Cys Phe Val Thr Thr Thr Gly Ser
100 105 110
Asn Asn Pro Ala Phe Val Ser Arg Val Gln Thr Leu Ile Ala Val Leu
115 120 125
Ser Gln Ser Ser Ser Asn Ala Ile Ser Gly Ala Thr Gly Gly Ser Ala
130 135 140
Phe Ala Gln Ser Gln Ala Phe Gln Gln Ser Ala Ser Gln Ser Ala Gly
145 150 155 160
Leu
<210> 4
<211> 139
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Gly Ile Ser Val Gly Val Pro Gly Tyr Leu Arg Thr Pro Ser Ser Thr
1 5 10 15
Ile Leu Ala Pro Ser Asn Ala Gln Ile Ile Ser Leu Gly Leu Gln Thr
20 25 30
Thr Leu Ala Pro Val Leu Ser Ser Ser Gly Leu Ser Ser Ala Ser Ala
35 40 45
Ser Ala Arg Val Ser Ser Leu Ala Gln Ser Leu Ala Ser Ala Leu Ser
50 55 60
Thr Ser Arg Gly Thr Leu Ser Leu Ser Thr Phe Leu Asn Leu Leu Ser
65 70 75 80
Ser Ile Ser Ser Glu Ile Arg Ala Ser Thr Ser Leu Asp Gly Thr Gln
85 90 95
Ala Thr Val Glu Val Leu Leu Glu Ala Leu Ala Ala Leu Leu Gln Val
100 105 110
Ile Asn Gly Ala Gln Ile Thr Asp Val Asn Val Ser Ser Val Pro Ser
115 120 125
Val Asn Ala Ala Leu Val Ser Ala Leu Val Ala
130 135
<210> 5
<211> 142
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Asn Leu Ser Ile Gly Asp Thr Thr Ser Ile Ile Gln Leu Phe Lys Asn
1 5 10 15
Phe Thr Gly Pro Pro Ser Val Ala Thr Phe Ile Ser Asn Phe His Ser
20 25 30
Ile Val Gln Ser Ser Lys Thr Leu Leu Asn Leu Phe Asp Val Ala Glu
35 40 45
Glu Asn Pro Leu Glu Phe Ala Lys Cys Met Tyr Glu Leu Val Leu Lys
50 55 60
Ser Ala Asn Ser Leu Gly Val Leu Asn Pro His Leu Ile Ala Asn Asn
65 70 75 80
Ile Tyr Gln Ser Val Val Ser Asn Leu Asp Ile Leu His Ser Ser Ala
85 90 95
Met Val Asn Leu Tyr Ala Asn Ala Met Ala Gly Ser Leu Phe Leu Glu
100 105 110
Gly Ile Leu Asn Ser Asp Asn Ala Ala Thr Leu Ala Lys Lys Cys Ala
115 120 125
Asn Asp Met Glu Ala Phe Ala Lys Lys Met Val Glu Ile Gly
130 135 140
<210> 6
<211> 642
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Gln Ala Ile Ser Val Ala Thr Ser Val Pro Ser Val Phe Ser Ser Pro
1 5 10 15
Ser Leu Ala Ser Gly Phe Leu Gly Cys Leu Thr Thr Gly Ile Gly Gln
20 25 30
Ser Pro Asp Phe Pro Phe Gln Glu Gln Gln Asp Leu Asp Asp Leu Ala
35 40 45
Gln Val Ile Leu Ser Ala Val Thr Ser Asn Thr Asp Thr Ser Lys Ser
50 55 60
Ala Arg Ala Gln Ala Leu Ser Thr Ala Leu Ala Ser Ser Leu Ala Asp
65 70 75 80
Leu Leu Ile Ser Glu Ser Ser Gly Ser Ser Tyr Gln Thr Gln Ile Ser
85 90 95
Ala Leu Thr Asn Ile Leu Ser Asp Cys Phe Val Thr Thr Thr Gly Ser
100 105 110
Asn Asn Pro Ala Phe Val Ser Arg Val Gln Thr Leu Ile Ala Val Leu
115 120 125
Ser Gln Ser Ser Ser Asn Ala Ile Ser Gly Ala Thr Gly Gly Ser Ala
130 135 140
Phe Ala Gln Ser Gln Ala Phe Gln Gln Ser Ala Ser Gln Ser Ala Gly
145 150 155 160
Leu Ser Ala Ser Arg Ala Gly Ser Thr Ser Ser Ser Thr Thr Thr Thr
165 170 175
Thr Ser Gly Ala Thr Ser Gln Ala Ala Ser Gln Ser Ala Ser Ser Ser
180 185 190
Tyr Ser Ser Ala Phe Ala Gln Ala Ala Ser Ser Ala Leu Ala Thr Ser
195 200 205
Ser Ala Ile Ser Arg Ala Phe Ala Ser Val Ser Ser Ala Ser Ala Ala
210 215 220
Ser Ser Leu Ala Tyr Asn Ile Gly Leu Ser Ala Ala Arg Ser Leu Gly
225 230 235 240
Ile Ala Ser Asp Thr Ala Leu Ala Gly Ala Leu Ala Gln Ala Val Gly
245 250 255
Gly Val Gly Ala Gly Ala Ser Ala Ser Ala Tyr Ala Asn Ala Ile Ala
260 265 270
Arg Ala Ala Gly Gln Phe Leu Ala Thr Gln Gly Val Leu Asn Ala Gly
275 280 285
Asn Ala Ser Ala Leu Ala Gly Ser Phe Ala Arg Ala Leu Ser Ala Ser
290 295 300
Ala Glu Ser Gln Ser Phe Ala Gln Ser Gln Ala Tyr Gln Gln Ala Ser
305 310 315 320
Ala Phe Gln Gln Ala Ala Ala Gln Ser Ala Ala Gln Ser Ala Ser Arg
325 330 335
Ala Gly Ser Thr Ser Ser Ser Thr Thr Thr Thr Thr Ser Gly Ala Thr
340 345 350
Ser Gln Ala Ala Ser Gln Ser Ala Ser Ser Ser Tyr Ser Ser Ala Phe
355 360 365
Ala Gln Ala Ala Ser Ser Ser Leu Ala Thr Ser Ser Ala Ile Ser Arg
370 375 380
Ala Phe Ala Ser Val Ser Ser Ala Ser Ala Ala Ser Ser Leu Ala Tyr
385 390 395 400
Asn Ile Gly Leu Ser Ala Ala Arg Ser Leu Gly Ile Ala Ser Asp Thr
405 410 415
Ala Leu Ala Gly Ala Leu Ala Gln Ala Val Gly Gly Val Gly Ala Gly
420 425 430
Ala Ser Ala Ser Ala Tyr Ala Asn Ala Ile Ala Arg Ala Ala Gly Gln
435 440 445
Phe Leu Ala Thr Gln Gly Val Leu Asn Ala Val Asn Ala Ser Ser Leu
450 455 460
Gly Ser Ala Leu Ala Asn Ala Leu Ser Asp Ser Ala Ala Asn Ser Ala
465 470 475 480
Val Ser Gly Asn Tyr Leu Gly Val Ser Gln Asn Phe Gly Arg Ile Ala
485 490 495
Pro Val Thr Gly Gly Thr Ala Gly Ile Ser Val Gly Val Pro Gly Tyr
500 505 510
Leu Arg Thr Pro Ser Ser Thr Ile Leu Ala Pro Ser Asn Ala Gln Ile
515 520 525
Ile Ser Leu Gly Leu Gln Thr Thr Leu Ala Pro Val Leu Ser Ser Ser
530 535 540
Gly Leu Ser Ser Ala Ser Ala Ser Ala Arg Val Ser Ser Leu Ala Gln
545 550 555 560
Ser Leu Ala Ser Ala Leu Ser Thr Ser Arg Gly Thr Leu Ser Leu Ser
565 570 575
Thr Phe Leu Asn Leu Leu Ser Ser Ile Ser Ser Glu Ile Arg Ala Ser
580 585 590
Thr Ser Leu Asp Gly Thr Gln Ala Thr Val Glu Val Leu Leu Glu Ala
595 600 605
Leu Ala Ala Leu Leu Gln Val Ile Asn Gly Ala Gln Ile Thr Asp Val
610 615 620
Asn Val Ser Ser Val Pro Ser Val Asn Ala Ala Leu Val Ser Ala Leu
625 630 635 640
Val Ala

Claims (4)

1. A recombinant spider ootheca silk protein compound comprises a recombinant ootheca silk protein monomer I and a monomer II, wherein the molecular framework of the recombinant ootheca silk protein monomer I is NTD- (RP 1) m -(RP2) n -CTD, wherein the integer m represents the repetition number of TuSp1-RP1 in TuSp 1-type repeat domain, n represents the repetition number of TuSp1-RP2 in type ii repeat domain, and m and n both range from 1 to 40; the molecular framework of the recombinant egg sheath silk protein monomer II is (RP) q Wherein, the integer q represents the repetition times of TuSp2-RP in the TuSp2 repetitive structural domain, and the value range is 1-40; the single TuSp1-RP1 amino acid sequence is shown as SEQ ID NO. 1, and the single TuSp1-RP2 amino acid sequence is shown as SEQ ID NO. 2; the recombinant ootheca silk protein monomer I also comprises a TuSp1 amino terminal non-repetitive structural domain TuSp1-NTD and a TuSp1 carboxyl terminal non-repetitive structural domain TuSp1-CTD; the TuSp1-NTD amino acid sequence is shown in SEQ ID NO. 3; the amino acid sequence of TuSp1-CTD is shown in SEQ ID NO. 4; the single TuSp2-RP amino acid sequence is shown in SEQ ID NO 5.
2. The recombinant spider ootheca silk protein complex of claim 1, characterized by NTD- (RP 1) m -(RP2) n -the sum of m and n in the molecular framework of CTD is less than or equal to 40.
3. The recombinant spider ootheca silk protein complex of claim 1, wherein each domain has a serine content of 10% or more.
4. The method for preparing the artificial synthetic spider ootheca silk by using the recombinant spider ootheca silk protein compound of claim 1 is characterized in that firstly, two types of recombinant spider ootheca silk protein expression plasmids are constructed, recombinant spider silk proteins are respectively overexpressed in escherichia coli, and the two recombinant spider silk proteins are mixed after expression and purification to form a compound, wherein the molar ratio of a recombinant ootheca silk protein monomer I to a monomer II is 3-25; the above complex was freeze-dried and dissolved in hexafluoroisopropanol, and then wet-spun in a coagulation bath containing zinc chloride and ferric chloride.
CN201910884960.1A 2019-09-19 2019-09-19 Recombinant spider ootheca silk protein compound and artificial ootheca silk generated by same Active CN110551194B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910884960.1A CN110551194B (en) 2019-09-19 2019-09-19 Recombinant spider ootheca silk protein compound and artificial ootheca silk generated by same

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910884960.1A CN110551194B (en) 2019-09-19 2019-09-19 Recombinant spider ootheca silk protein compound and artificial ootheca silk generated by same

Publications (2)

Publication Number Publication Date
CN110551194A CN110551194A (en) 2019-12-10
CN110551194B true CN110551194B (en) 2023-03-21

Family

ID=68740770

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910884960.1A Active CN110551194B (en) 2019-09-19 2019-09-19 Recombinant spider ootheca silk protein compound and artificial ootheca silk generated by same

Country Status (1)

Country Link
CN (1) CN110551194B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4388256A (en) * 1978-11-24 1983-06-14 Masamichi Ishida Process for manufacturing regenerated cellulose hollow fiber
WO1991016351A1 (en) * 1990-04-19 1991-10-31 The United States Of America, Secretary Of The Army, The Pentagon Recombinant spider silk proteins through genetic engineering
CN109912720A (en) * 2019-03-14 2019-06-21 天津大学 A kind of the design synthetic method and spinning of spider's thread protein

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010123450A1 (en) * 2009-04-22 2010-10-28 Spiber Technologies Ab Method of producing polymers of spider silk proteins

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4388256A (en) * 1978-11-24 1983-06-14 Masamichi Ishida Process for manufacturing regenerated cellulose hollow fiber
WO1991016351A1 (en) * 1990-04-19 1991-10-31 The United States Of America, Secretary Of The Army, The Pentagon Recombinant spider silk proteins through genetic engineering
CN109912720A (en) * 2019-03-14 2019-06-21 天津大学 A kind of the design synthetic method and spinning of spider's thread protein

Also Published As

Publication number Publication date
CN110551194A (en) 2019-12-10

Similar Documents

Publication Publication Date Title
EP1919938B1 (en) Modified spider silk proteins
JP5128943B2 (en) Recombinant spider silk protein
US9051453B2 (en) Polypeptide solution, artificial polypeptide fiber production method and polypeptide purification method using same
US20120231499A1 (en) High-molecular-weight recombinant silk or silk-like protein and micro- or nano-sized spider silk or silk-like fiber produced therefrom
CA2635660C (en) Spider silk proteins and methods for producing spider silk proteins
Xu et al. Recombinant minimalist spider wrapping silk proteins capable of native-like fiber formation
ES2887366T3 (en) Methods and compositions for synthesizing improved silk fibers
US7521228B2 (en) Spider silk protein encoding nucleic acids, polypeptides, antibodies and methods of use thereof
Chen et al. The spinning processes for spider silk
US20210317173A1 (en) Compositions and methods for fabricating synthetic dragline spider silk
CN109890880A (en) Composite molding composition and preparation method thereof containing fibroin sample protein
Zhu et al. Tensile properties of synthetic pyriform spider silk fibers depend on the number of repetitive units as well as the presence of N-and C-terminal domains
CN108456246B (en) Recombinant spider silk protein, preparation method and application thereof
US9732125B2 (en) Polypeptide particle and method for producing same
CN111454370A (en) Chimeric protein and preparation method and application thereof
JP2018531040A6 (en) Compositions and methods for making synthetic dragline spider silks
Xu et al. Structural characterization and mechanical properties of chimeric Masp1/Flag minispidroins
US8461301B2 (en) Synthetic dragline spider silk-like proteins
CN107805283B (en) Cobweb-like protein and biosynthesis method thereof
CN110551194B (en) Recombinant spider ootheca silk protein compound and artificial ootheca silk generated by same
JP2012055269A (en) Nucleic acid, protein encoded by the nucleic acid, recombinant organism having the nucleic acid introduced therein, and protein produced by the recombinant organism
US20150057435A1 (en) Recombinant silk protein derived from sea anemones, method for manufacturing same, and composition for manufacturing silk fibers including same
CN114685686B (en) Fusion protein and application thereof in preparation of biological protein fiber
CN107849100B (en) Polar solvent solution and method for producing same
CN110117328B (en) Recombinant spider silk protein and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant