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

Abstract

The invention relates to a recombinant spider egg sheath silk protein complex and the artificial egg sheath silk produced therefrom; the complex is composed of a molecular framework of NTD-(RP1) _m- (RP2) _n -CTD and a molecular framework of (RP) _q ; construction Two types of recombinant spidroin protein expression plasmids, and overexpressed recombinant spidroin protein in Escherichia coli respectively, the molar ratio of recombinant ootheca silk protein monomer I:monomer II is 3-25:1; the above complex is freeze-dried , and dissolved in hexafluoroisopropanol to form a spidroin solution; then wet spinning was carried out in a coagulation bath containing zinc chloride and ferric chloride. The tensile strength of the artificial egg sheath silk produced by the recombinant egg sheath silk protein compound in the invention is increased by about 35% on average, which exceeds the strength of the natural egg sheath silk of the same type.

Description

Recombinant spider egg sheath silk protein complex and its produced artificial egg sheath silk

technical field

The invention relates to a recombinant spider egg sheath silk protein complex and an artificial egg sheath silk produced therefrom; in particular, a recombinant spider egg sheath silk protein complex and a method for manufacturing high-strength artificial egg sheath silk using the complex.

Background technique

Spider silk has excellent mechanical properties and potential wide application prospects, and its applicable fields include textile industry, optics, environmental engineering, biomedicine and military industry, etc., such as microsurgical sutures, tissue engineering scaffolds, carriers for drug delivery, hemostasis Bandages, bulletproof materials, etc. These materials must have specific physical properties, such as certain strength, toughness, elasticity, biodegradability, and biocompatibility, so spider silk that meets these conditions has received a lot of attention (Vollrath, F. & Knight, D.P., Nature, 410:541, 2001). However, spiders have the characteristics of cannibalism and cannot be raised on a large scale. Therefore, people have studied the production mechanism of spider silk in depth, tried to produce artificial spider silk protein through genetic engineering, protein engineering or synthetic biology, and artificially spun artificial spider silk in vitro by simulating the spider's spinner and silk-forming conditions (Lazaris , A. et al, Science, 2002, 295:472; Teule, F. et al, Nat. Protoc., 2009, 4: 341; Scheller, J. et al, Nat. Biotechnol., 2001, 19: 573) .

The spidroin protein that constitutes spider silk is composed of multiple repeating domains in the middle and non-repeating domains at both ends, and has a high molecular weight (MW), ranging from ~200kDa to ~1380kDa (Chen G. et al. PLOS One, 2012,7:e52293; Wen R.et al.Int. J.Biol.Macromol.,2018,117:1352; Stellwagen S.D.etal.G3:Genes Genom.Genet.,2019,9:1909); Moreover, the same type The spider silk of the spider is usually composed of a variety of spidroin proteins (Hu X. et al. Biochemistry, 2006, 45: 3506; Huang W. et al. Sci. Rep., 2017, 7: 13354; Garb J. E. et al. Commun. Biol., 2019, 2:275). This means that the size and species composition of spidroin proteins are key determinants of the excellent mechanical properties of spider silk. At present, researchers mainly produce single recombinant spidroin protein through expression systems such as Escherichia coli, yeast, plant cells, insect cells and mammalian cells (Hauptmann V.etal.Transgenic Res.,2013, 22:369; Lin Z.et al .Adv.Mater.,2013,25:1216; Lazaris,A.Science,2002,295:472;Huemmerich D.et al.Curr.Biol.,2004,14:2070;JanssonR.et al.Biotechnol.J. , 2016, 11:687). However, the expression of exogenous proteins with high molecular weight (for example, more than 200kD) in E. coli will lead to a serious decrease in expression. In order to solve the problem of large-molecular-weight spidroin expression in vitro, researchers have tried to use plant expression systems to obtain multi-polymer recombinant silk protein mixtures with 2 to 10 structural domains, but they have not been able to obtain uniform high-molecular-weight recombinant silk proteins (Hauptmann V . et al. Transgenic Res., 2013, 22:369).

PCT Patent Document No. WO2007/078239 discloses a method of expressing a high-molecular-weight recombinant dragline protein (about 285kDa) using a modified Escherichia coli BL21 (DE3) strain, and using the single recombinant protein to artificially spin an artificial protein with better mechanical properties Silk. But its breaking strength is still not as good as that of natural draw wire.

Another approach is to covalently link different spidroin proteins together through disulfide bonds to increase the molecular weight. The use of non-specific linkage can only produce heterogeneous polymers, and the content of large molecular weight polymers is too low, so the mechanical properties of rayon fibers are only slightly improved (Grip S.et al.Protein Sci.,2010,18:1012 ). However, it can produce uniform high molecular weight recombinant silk protein (about 378kDa) by connecting specifically through the terminal domain, and the artificial silk spun from it can be comparable to the corresponding natural spider silk in terms of tensile strength (Lin Z. et al . Adv. Mater., 2013, 25:1216). Recently, people have been able to use an ultra-high molecular weight recombinant dragline protein (about 556 kDa) formed by intein cleavage ligation to prepare high-performance artificial draglines, which have been improved in tensile strength and elongation. It is close to the strength of natural drawn silk (Bowen C.H. et al. Biomacromolecules, 2018, 19:3853). However, this kind of recombinant silk must be prepared using spinning solutions that are highly toxic to the body, such as methanol, etc., so it is difficult to use in industrial scale production superior. In conclusion, there are great difficulties in the mass production of artificial silk spun from high-molecular-weight spider silk proteins; in addition, the production of artificial spider silk mainly relies on a single recombinant silk protein, which may also be because people cannot prepare it under mild or weak toxicity conditions. One of the main reasons why rayon has excellent mechanical properties.

According to existing studies, the spidroin proteins isolated from spider egg sheath silk mainly include TuSp ( Tu buliform sp idroin) (Zhao A. et al. Biochemistry, 2006, 45: 3348; Huang W. et al. Biochimie, 2006 ,88:849). In the egg sheath silk of the black widow spider Latrodectus hesperus, there are two other different silk proteins: ECP-1 ( E gg case p protein-1) and ECP-2 (Hu X. et al. Biochemistry, 2006 , 45:3506). In the golden orb-web spider Nephila antipodiana, egg sheath silk has been identified to contain at least two silk proteins, TuSp1 and TuSp2 (Huang W. et al. Sci. Rep., 2017, 7: 13354). TuSp1 is the core protein of egg sheath filament, and it is also the main component. Its full-length molecular weight is about 360kDa, but its complete sequence is still unknown. Studies on the amino acid sequence and advanced structure of silk protein show that TuSp1 contains multiple conserved repeat domains (TuSp1 Repetitive domain, TuSp1-RP), as well as an amino-terminal (TuSp1 N-terminal domain, TuSp1-NTD) and a carboxy-terminal non-repeating structure Domain (TuSp1 C-terminal domain, TuSp1-CTD) (Lin Z. et al. Proc. Natl. Acad. Sci. USA, 2009, 106:8906). TuSp1-RP can be further divided into two types, namely the type-1 repeat domain (TuSp1-RP1) and the type-2 repeat domain (TuSp1-RP2); the content of alanine and serine in the repeat sequence is relatively high (about 40- 50%), which is different from traction filaments and small ampullate filaments which are rich in alanine and glycine. Another egg sheath silk protein, TuSp2, is a non-main component of egg sheath silk. Although it also contains multiple repeat domains (TuSp2-RP), it does not have the typical silk protein carboxy-terminal non-repeat domain, and its The sequence of the amino terminus is also unknown. Its structure is rich in alanine, serine, asparagine and leucine (total about 45%). Therefore, in order to obtain artificial egg sheath silk with excellent mechanical properties, the present invention designs a compound containing two different types of recombinant egg sheath silk proteins based on the current research on the molecular structure of the golden orb-web spider egg sheath silk protein, and artificially The artificial egg sheath protein yarn with better mechanical properties than the existing artificial egg sheath silk is produced by weaving, and it also surpasses the natural egg sheath silk in tensile strength.

Contents of the invention

The first object of the present invention is to provide a recombinant silk protein complex for producing artificial spider egg sheath silk. The second purpose of the present invention is to use the above-mentioned recombinant silk protein complex as a raw material to form filaments in a low-toxicity coagulation solution to obtain a new type of artificial egg sheath silk whose strength is significantly improved compared with that of a single recombinant protein.

Technical scheme of the present invention is as follows:

A recombinant spider egg sheath silk protein complex; composed of recombinant egg sheath silk protein monomer I and monomer II, wherein the molecular framework of the recombinant egg sheath silk protein monomer I is NTD-(RP1) _m -(RP2 ) _n -CTD, wherein, the integer m represents the repeat number of TuSp1-type repeat domain TuSp1-RP1, n represents the repeat number of type-2 repeat domain TuSp1-RP2, and the value range of m and n is 1-40; the recombinant egg The molecular structure of the sheath fibroin monomer II is (RP) _q , where the integer q represents the repeat number of the TuSp2 repeat domain TuSp2-RP, and the value ranges from 1 to 40.

The recombinant spider egg sheath silk protein complex; its single TuSp1-RP1 amino acid sequence is shown in SEQ ID NO: 1, and the single TuSp1-RP2 amino acid sequence is shown in SEQ ID NO: 2; the recombinant egg sheath silk protein single Body I also includes a TuSp1 amino-terminal non-repeating domain TuSp1-NTD, the amino acid sequence of which is shown in SEQ ID NO: 3; and a TuSp1 carboxy-terminal non-repeating domain TuSp1-CTD, the amino acid sequence of which is shown in SEQ ID NO: 4 Show.

The recombinant spider egg sheath silk protein complex; its single TuSp2-RP amino acid sequence is shown in SEQ ID NO:5.

The recombinant spider egg sheath silk protein complex; its molecular structure is that the sum of m and n in NTD-(RP1) _m -(RP2) _n -CTD is less than or equal to 40.

The recombinant spider egg sheath silk protein complex; each structural domain has a structure with a serine content of more than 10%.

The method for artificially synthesizing spider egg sheath silk using the recombinant spider egg sheath silk protein complex of the present invention; first, construct two types of recombinant spidroin protein expression plasmids, and respectively overexpress the recombinant spidroin protein in Escherichia coli. After purification, the two recombinant spidroin proteins are mixed to form a complex. In the complex, the molar ratio of recombinant ovaginin monomer I:monomer II is 3 to 25:1; the above complex is freeze-dried and used Hexafluoroisopropanol is dissolved and then wet-spun in a coagulation bath containing zinc chloride and ferric chloride.

The structural domain sequence description involved in the present invention is as shown in Table 1:

Table 1

Compared with the existing single-type artificial egg sheath silk, the beneficial effect of the present invention is that the mechanical properties of the artificial egg sheath silk manufactured by using the recombinant egg sheath silk protein complex are greatly improved. When the molar ratio of recombinant egg sheath protein monomer I:monomer II is 6:1, the tensile strength is increased by about 35% on average, exceeding the strength of the same type of natural egg sheath silk. In addition, the present invention uses inorganic zinc/iron salt solution as a coagulation bath and alcohol solution as a post-treatment reagent in the process of making artificial egg sheath silk, compared with highly toxic organic reagents such as isopropanol and methanol used in previous studies It is more green and environmentally friendly, which provides a further possibility for the industrial scale production of artificial egg sheath silk.

Description of drawings

Figure 1 shows a schematic diagram of the molecular architecture of the recombinant spidroin proteins TuSp1-N2RPC and TuSp2-RP.

Fig. 2 shows the expression system used for expressing the recombinant vitellin monomer and the vitellin recombination module on the expression vector.

Figure 3 is the SDS-PAGE analysis chart of purified recombinant spidroin proteins TuSp1-N2RPC and TuSp2-RP, wherein M is the protein molecular weight standard; lane 1 is the purified sample of TuSp1-N2RPC; lane 2 is the purified sample of TuSp2-RP.

Figure 4 is a schematic diagram of spinning equipment.

Figure 5 is conventional optical microscope (a) and polarizing microscope (b) images of artificial egg sheath filaments formed by TuSp1-N2RPC:TuSp2-RP complex (molar ratio 6:1), the scale bar is 50 μm.

Fig. 6 is a comparison diagram of stress curves of artificial egg sheath wire. i, control group TuSp1-N2RPC; ii～v, TuSp1-N2RPC:TuSp2-RP, the molar ratios were 3:1(ii), 6:1(iii), 12:1(iv) and 25:1(v) ).

Fig. 7 is a comparison chart of tensile strength (a), breaking energy (b) and Young's modulus (c) of artificial egg sheath silk. i, control group TuSp1-N2RPC; ii-v, TuSp1-N2RPC:TuSp2-RP, molar ratios were 3:1(ii), 6:1(iii), 12:1(iv) and 25:1(v ).

Detailed ways

The specific embodiment of the present invention is described in detail below with example, the compound that is used to manufacture the artificially synthesized spider egg sheath silk in this example includes two kinds of recombinant egg sheath silk proteins, namely TuSp1-N2RPC (recombinant egg sheath silk protein monomer I ) and TuSp2-RP (recombinant vitrein monomer II), their molecular structures are NTD-(RP1) ₁ -(RP2) ₁ -CTD and (RP) ₁ respectively (as shown in Figure 1). Among them, TuSp1-N2RPC is a recombinant spidroin protein from TuSp1, consisting of four different TuSp1 domains, namely a TuSp1-RP1 (corresponding to the sequence number SEQ ID NO: 1), a TuSp1-RP2 (corresponding to the sequence number SEQ ID NO:2), a TuSp1-NTD (corresponding to the sequence number SEQ ID NO:3) and a TuSp1-CTD (corresponding to the sequence number SEQ ID NO:4). The serine contents of the above four domains are 23%, 23%, 21% and 20%, respectively. TuSp2-RP is a recombinant spidroin protein of TuSp2, which only contains one TuSp2-RP (corresponding to the sequence number SEQ ID NO: 5), and its serine content is 11%. The specific embodiment that artificial spider egg sheath silk is prepared in this example is as follows:

Example 1: Construction of Escherichia coli expression plasmids for recombinant spidroin proteins TuSp1-N2RPC and TuSp2-RP.

1.1 Construction of recombinant egg sheath protein expression plasmid pTuSp1-N2RPC.

Acquisition of TuSp1-N2RPC target gene. Based on the previous research on the molecular structure and gene sequence of the egg sheath silk protein TuSp1 of the golden orb-web spider (Lin Z. et al. Proc. Natl. Acad. Sci. USA, 2009, 106:8906), four genes were obtained by gene synthesis Modules, respectively encoding TuSp1-RP1, TuSp1-RP2, TuSp1-NTD and TuSp1-CTD, and then constructing TuSp1-N2RPC containing TuSp1-N2 through head-to-tail circular connection (Lin Z.et al.Adv.Mater., 2013,25:1216) The expression plasmid pTuSp1-N2RPC of the recombination module (as shown in FIG. 2 ). Finally, the constructed recombinant plasmid was confirmed by restriction endonuclease digestion and base sequence analysis.

1.2 Construction of recombinant egg sheath protein expression plasmid pTuSp2-RP.

Acquisition of TuSp2-RP target gene. Based on the previous study on the molecular structure of the egg sheath silk protein TuSp2 of the golden orb-web spider (Huang W. Sci.Rep.2017, 7:13354), the gene encoding TuSp2-RP was obtained by gene synthesis, and two genes, BamH I and Xho I, were used to An expression plasmid pTuSp2-RP containing the TuSp2-RP gene was constructed with restriction endonucleases (as shown in FIG. 2 ). Finally, the constructed recombinant plasmid was confirmed by restriction endonuclease digestion and base sequence analysis.

The bacterial strains, plasmid vectors, antibiotics and medium involved in this embodiment are: cloning host E.coli DH5α, expression plasmid pET32a+ (without Trx and S tags), ampicillin, LB medium (10g/L tryptone, 5g/L 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 spidroin protein.

The two recombinant spidroin proteins in the complex of the present invention are respectively expressed in an Escherichia coli expression system and purified by nickel column affinity chromatography.

2.1 Expression and purification of TuSp1-N2RPC recombinant spidroin protein

(1) Preparation of seed solution. The recombinant plasmid containing pTuSp1-N2RPC was transformed into Escherichia coli E.coli BL21(DE3)pLysS to form an expression strain. Inoculate the above-mentioned monoclonal strain into TB liquid medium (12g/L tryptone, 24g/L yeast powder, 4mL/L glycerol and 50mM phosphate buffer) with a pH of 6.8, add 100μg/ml ampicillin and 34μg/L ml chloramphenicol, cultured on a shaker at 220 rpm at 37°C for 10-12 hours.

(2) Induced expression. Transfer the prepared seed solution to fresh TB liquid medium with a pH of 6.8, add 100 μg/ml ampicillin and 34 μg/ml chloramphenicol, culture at 37°C, 220rpm shaker until the _OD600 is 1.2-2.8, Add isopropyl-β-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM to induce expression at 16-25° C. for 12-18 hours, and collect the bacterial cells.

(3) Protein purification. The collected TuSp1-N2RPC cells were resuspended in sterile water, and ultrasonically disrupted in an ice bath using a sonicator with a power of 270W for 20 to 45 minutes. Centrifuge to collect the ultrasonic lysed supernatant and precipitate, denature the precipitate with 8M urea until uniform and clear, and then centrifuge again to collect the denatured supernatant. The denatured supernatant containing TuSp1-N2RPC was purified using a nickel column, collected and dialyzed. The analysis results of the obtained recombinant silk protein TuSp1-N2RPC are shown in lane 1 in FIG. 3 , and the purity is about 90%.

2.2 Expression and purification of TuSp2-RP recombinant spidroin protein

(1) Preparation of seed solution. The recombinant plasmid containing pTuSp2-RP was transformed into Escherichia coli E. coli BL21(DE3)pLysS to form an expression strain. Inoculate the above monoclonal strain into LB medium (10g/L tryptone, 5g/L yeast powder and 10g/L sodium chloride), add 100μg/ml ampicillin and 34μg/ml chloramphenicol, 37°C, 220rpm Incubate on a shaker for 10-12 hours.

(2) Induced expression. Transfer the prepared seed solution to fresh LB medium, add 100 μg/ml ampicillin and 34 μg/ml chloramphenicol, culture at 37°C, shaker at 180 rpm until the OD ₆₀₀ is 0.5-1.0, and the final concentration is 0.1 mM The expression of IPTG was induced at 16-25°C for 12-18 hours, and the cells were collected.

(3) Protein purification. After the collected TuSp2-RP cells were resuspended with Lysis Buffer containing Tris, the cells were disrupted by a low-temperature ultra-high pressure continuous flow cell disruptor, the supernatant was collected by centrifugation, and purified, collected and dialyzed using a nickel column. The analysis results of the obtained recombinant silk protein TuSp2-RP are shown in lane 2 of FIG. 3 , and the purity is about 90%.

Example 3 Preparation and spinning process of artificial egg sheath silk spinning stock solution.

3.1 Preparation of spinning dope.

The purified recombinant spidroin proteins TuSp1-N2RPC and TuSp2-RP were lyophilized for 48 hours. Mix TuSp1-N2RPC and TuSp2-RP in a certain ratio to form a complex. In the above complexes, the molar ratio of TuSp1-N2RPC to TuSp2-RP is minimum 3:1 and maximum 25:1. The complex is dissolved in hexafluoroisopropanol at room temperature for 8 hours to form a uniform recombined spidroin solution with a mass percentage of 10-15%, which is the spinning stock solution. The spinning dope of the control group contained only a single recombinant spidroin protein TuSp1-N2RPC, with a mass percentage of 10-15%.

3.2 Preparation of primary artificial spider egg sheath silk fibers.

First, an aqueous solution containing 100 mM zinc chloride and 2 mM ferric chloride was prepared as a coagulation bath for synthesizing spider egg sheath silk at a temperature of 25°C. Secondly, 500 μl of the prepared spinning stock solution was transferred to a 1 ml syringe, and the silk outlet with a diameter of about 127 μm was immersed in the coagulation bath, and the silk protein complex became silk in the coagulation bath. Finally, the nascent artificial spider egg sheath silk formed in the coagulation bath is wound on the reel at a uniform speed. The schematic diagram of the spinning equipment is shown in Fig. 4.

3.3 Post-processing of artificial spider egg sheath silk.

The nascent artificial spider egg sheath silk fiber has irregular internal molecular structure and poor mechanical properties, so it needs to undergo certain post-treatment to enhance its mechanical properties. In the present invention, the newborn artificial spider egg sheath silk fiber is immersed in 80-90% alcohol solution and stretched 4-5 times at a constant speed of 3-4 mm/min, taken out and fixed, and dried at room temperature to obtain the artificial spider Egg sheath silk fibers with a diameter of 5-8 μm. A representative post-processed artificial egg sheath silk fiber image is shown in Figure 5; among them, the polarized light micrograph shows that the artificial silk has obvious birefringence, which indicates that the silk protein molecules are well aligned along specific directions. In this example, although the composites had different monomer ratios, no significant differences were seen in the photomicrographs of the silk fibers formed.

3.4 Mechanical property test of synthetic spider egg sheath silk.

Take the dried artificial egg sheath fiber, fix it on a fixture, use an optical microscope to observe its surface features and measure its diameter. We selected samples (n=3) with a smooth surface and a uniform diameter of 7-10 μm at a relative humidity of 40% and room temperature, and tested the mechanical properties on a universal tensile testing machine (E42.503, MTS) with a sensor with a rated load of 5N. The test: the gauge length is 20mm, and the tensile speed is 10mm/min. Figure 6 shows the stress-strain curves of two types of artificial egg sheath wires.

All the measurement results are shown in Figure 7. Compared with the control group, that is, the artificial egg sheath silk TuSp1-N2RPC containing only a single recombinant silk protein, the artificial composite egg sheath silk TuSp1-N2RPC:TuSp2-RP (molar ratio 6:1) Mechanical properties (comprising tensile strength, breaking energy and Young's modulus) have been improved; Wherein, tensile strength is 437 ± 43MPa, has improved about 35% than the intensity of above-mentioned control group, is the currently known artificial ootheca Highest tensile strength among silks.

In summary, the artificial spider egg sheath silk of the present invention uses a complex formed by two recombinant spider egg sheath silk proteins to prepare spinning stock solution, uses a low-toxic and environmentally friendly inorganic salt solution as a coagulation bath, and wet spinning Silk way to obtain artificial spider egg sheath silk. Compared with the artificial spider silk formed by single recombinant egg sheath silk protein, its mechanical properties are significantly improved. The design of the ootheca fibroin in the present invention is based on the current research on the structure and function of the natural ootheca fibroin to select and combine the necessary functional modules. Since the two recombinant egg sheath proteins do not have high molecular weight, they can be highly expressed in the conventional E. coli expression system, and can be mixed in any proportion before spinning to form rayon with specific physical properties. As long as there is no contradiction, each technical feature of the above-mentioned embodiments should be considered as within the scope of the description. The above-mentioned embodiments only express the implementation of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. In addition, for researchers in the field, without departing from the concept of the present invention, some modifications and improvements can be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on 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 Thr Thr Ser Gly Ala Thr Ser Gln Ala Ala Ser Gln Ser Ala Ser Ser Ser Tyr Ser Ser Ala PheAla Gln Ala Ala Ser Ser Ala Leu Ala Thr Ser Ser Ser Ala Ile Ser Arg Ala Phe AlaSer Val Ser Ser Ala Ser Ala Ala Ser Ser Leu Ala Tyr Asn Ile Gly Leu Ser AlaAla Arg Ser Leu Gly Ile Ala Ser Asp Thr Ala Leu Ala Gly Ala Leu Ala Gln AlaVal Gly Gly Val Gly Ala Gly Ala Ser Ala Ser Ala Tyr Ala Asn Ala Ile Ala ArgAla Ala Gly Gln Phe Leu Ala Thr Gln Gly Val Leu Asn Ala Gly Asn Ala Ser AlaLeu Ala Gly Ser Phe Ala Arg Ala Leu Ser Ala Ser Ala Glu Ser Gln Ser Phe AlaGln 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 Thr Thr Ser Gly Ala Thr Ser Gln Ala Ala Ser Gln Ser Ala Ser Ser Ser Tyr Ser Ser Ala PheAla Gln Ala Ala Ser Ser Ser Ser Leu Ala Thr Ser Ser Ser Ala Ile Ser Arg Ala Phe AlaSer Val Ser Ser Ala Ser Ala Ala Ser Ser Leu Ala Tyr Asn Ile Gly Leu Ser AlaAla Arg Ser Leu Gly Ile Ala Ser Asp Thr Ala Leu Ala Gly Ala Leu Ala Gln AlaVal Gly Gly Val Gly Ala Gly Ala Ser Ala Ser Ala Tyr Ala Asn Ala Ile Ala ArgAla Ala Gly Gln Phe Leu Ala Thr Gln Gly Val Leu Asn Ala Val Asn Ala Ser SerLeu Gly Ser Ala Leu Ala Asn Ala Leu Ser Asp Ser Ala Ala Asn Ser Ala Val SerGly Asn Tyr Leu Gly Val Ser Gln Asn Phe Gly Arg Ile Ala Pro Val Thr Gly GlyThr Ala

SEQ ID NO:3

Gln Ala Ile Ser Val Ala Thr Ser Val Pro Ser Val Phe Ser Ser Pro SerLeu Ala Ser Gly Phe Leu Gly Cys Leu Thr Thr Gly Ile Gly Gln Ser Pro Asp PhePro Phe Gln Glu Gln Gln Asp Leu Asp Asp Leu Ala Gln Val Ile Leu Ser Ala ValThr Ser Asn Thr Asp Thr Ser Lys Ser Ala Arg Ala Gln Ala Leu Ser Thr Ala LeuAla Ser Ser Leu Ala Asp Leu Leu Ile Ser Glu Ser Ser Ser Gly Ser Ser Ser Tyr Gln ThrGln Ile Ser Ala Leu Thr Asn Ile Leu Ser Asp Cys Phe Val Thr Thr Thr Gly SerAsn Asn Pro Ala Phe Val Ser Arg Val Gln Thr Leu Ile Ala Val Leu Ser Gln SerSer Ser Asn Ala Ile Ser Gly Ala Thr Gly Gly Ser Ala Phe Ala Gln Ser Gln AlaPhe 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 IleLeu Ala Pro Ser Asn Ala Gln Ile Ile Ser Leu Gly Leu Gln Thr Thr Leu Ala ProVal Leu Ser Ser Ser Gly Leu Ser Ser Ala Ser Ala Ser Ala Arg Val Ser Ser LeuAla Gln Ser Leu Ala Ser Ala Leu Ser Thr Ser Arg Gly Thr Leu Ser Leu Ser ThrPhe Leu Asn Leu Leu Ser Ser Ile Ser Ser Glu Ile Arg Ala Ser Thr Ser Leu AspGly Thr Gln Ala Thr Val Glu Val Leu Leu Glu Ala Leu Ala Ala Leu Leu Gln ValIle Asn Gly Ala Gln Ile Thr Asp Val Asn Val Ser Ser Val Pro Ser Val Asn AlaAla 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 PheThr Gly Pro Pro Ser Val Ala Thr Phe Ile Ser Asn Phe His Ser Ile Val Gln SerSer Lys Thr Leu Leu Asn Leu Phe Asp Val Ala Glu Glu Asn Pro Leu Glu Phe AlaLys Cys Met Tyr Glu Leu Val Leu Lys Ser Ala Asn Ser Leu Gly Val Leu Asn ProHis Leu Ile Ala Asn Asn Ile Tyr Gln Ser Val Val Ser Asn Leu Asp Ile Leu HisSer Ser Ala Met Val Asn Leu Tyr Ala Asn Ala Met Ala Gly Ser Leu Phe Leu GluGly Ile Leu Asn Ser Asp Asn Ala Ala Thr Leu Ala Lys Lys Cys Ala Asn Asp MetGlu 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 SerLeu Ala Ser Gly Phe Leu Gly Cys Leu Thr Thr Gly Ile Gly Gln Ser Pro Asp PhePro Phe Gln Glu Gln Gln Asp Leu Asp Asp Leu Ala Gln Val Ile Leu Ser Ala ValThr Ser Asn Thr Asp Thr Ser Lys Ser Ala Arg Ala Gln Ala Leu Ser Thr Ala LeuAla Ser Ser Leu Ala Asp Leu Leu Ile Ser Glu Ser Ser Ser Gly Ser Ser Ser Tyr Gln ThrGln Ile Ser Ala Leu Thr Asn Ile Leu Ser Asp Cys Phe Val Thr Thr Thr Gly SerAsn Asn Pro Ala Phe Val Ser Arg Val Gln Thr Leu Ile Ala Val Leu Ser Gln SerSer Ser Asn Ala Ile Ser Gly Ala Thr Gly Gly Ser Ala Phe Ala Gln Ser Gln AlaPhe Gln Gln Ser Ala Ser Gln Ser Ala Gly Leu Ser Ala Ser Arg Ala ThrSer Ser Ser Thr Thr Thr Thr Thr Thr Thr Thr Ser Gly Ala Thr Ser Gln Ala Ala Ser Gln SerAla Ser Ser Ser Ser Tyr Ser Ser Ala Phe Ala Gln Ala Ala Ser Ser Ala Leu Ala ThrSer Ser Ala Ile Ser Arg Ala Phe Ala Ser Val Ser Ser Ala Ser Ala Ala Ser SerLeu Ala Tyr Asn Ile Gly Leu Ser Ala Ala Arg Ser Leu Gly Ile Ala Ser Asp ThrAla Leu Ala Gly Ala Leu Ala Gln Ala Val Gly Gly Val Gly Ala Gly Ala Ser AlaSer Ala Tyr Ala Asn Ala Ile Ala Arg Ala Ala Gly Gln Phe Leu Ala Thr Gln GlyVal Leu Asn Ala Gly Asn Ala Ser Ala Leu Ala Gly Ser Phe Ala Arg Ala Leu SerAla Ser Ala Glu Ser Gln Ser Phe Ala Gln Ser Gln Ala Tyr Gln Gln Ala Ser AlaPhe Gln Gln Ala Ala Ala Gln Ser Ala Ala Gln Ser Ala Ser Arg Ala Gly Ser ThrSer Ser Ser Thr Thr Thr Thr Thr Thr Thr Ser Ser Gly Ala Thr Ser Gln Ala Ala Ser Gln SerAla Ser Ser Ser Ser Tyr Ser Ser Ser Ala Phe Ala Gln Ala Ala Ser Ser Ser Leu Ala ThrSer Ser Ala Ile Ser Arg Ala Phe Ala Ser Val Ser Ser Ala Ser Ala Ala Ser SerLeu Ala Tyr Asn Ile Gly Leu Ser Ala Ala Arg Ser Leu Gly Ile Ala Ser Asp ThrAla Leu Ala Gly Ala Leu Ala Gln Ala Val Gly Gly Val Gly Ala Gly Ala Ser AlaSer Ala Tyr Ala Asn Ala Ile Ala Arg Ala Ala Gly Gln Phe Leu Ala Thr Gln GlyVal Leu Asn Ala Val Asn Ala Ser Ser Leu Gly Ser Ala Leu Ala Asn Ala Leu SerAsp Ser Ala Ala Asn Ser Ala Val Ser Gly Asn Tyr Leu Gly Val Ser Gln Asn PheGly Arg Ile Ala Pro Val Thr Gly Gly Thr Ala Gly Ile Ser Val Gly Val Pro GlyTyr Leu Arg Thr Pro Ser Ser Thr Ile Leu Ala Pro Ser Asn Ala Gln Ile Ile SerLeu Gly Leu Gln Thr Thr Leu Ala Pro Val Leu Ser Ser Ser Gly Leu Ser Ser AlaSer Ala Ser Ala Arg Val Ser Ser Leu Ala Gln Ser Leu Ala Ser Ala Leu Ser ThrSer Arg Gly Thr Leu Ser Leu Ser Thr Phe Leu Asn Leu Leu Ser Ser Ile Ser Ser SerGlu Ile Arg Ala Ser Thr Ser Leu Asp Gly Thr Gln Ala Thr Val Glu Val Leu LeuGlu Ala Leu Ala Ala Leu Leu Gln Val Ile Asn Gly Ala Gln Ile Thr Asp Val AsnVal Ser Ser Ser Val Pro Ser Val Asn Ala Ala Leu Val Ser Ala Leu Val Ala.

sequence listing

<110> Tianjin University

<120> Recombinant spider egg sheath silk protein complex and its produced artificial egg sheath silk

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 171

<212> PRT

<213> Artificial Sequence

<400> 1

Ser Ala Ser Arg Ala Gly Ser Thr Ser Ser Ser Ser Thr Thr Thr Thr Thr Thr Thr

1 5 10 15

Ser Gly Ala Thr Ser Gln Ala Ala Ser Gln Ser Ala Ser 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

<400> 2

Ser Ala Ser Arg Ala Gly Ser Thr Ser Ser Ser Ser Thr Thr Thr Thr Thr Thr Thr

1 5 10 15

Ser Gly Ala Thr Ser Gln Ala Ala Ser Gln Ser Ala Ser 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

<400> 3

Gln Ala Ile Ser Val Ala Thr Ser 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 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

<400> 4

Gly Ile Ser Val Gly Val Pro Gly Tyr Leu Arg Thr Pro Ser 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 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 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

<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

<400> 6

Gln Ala Ile Ser Val Ala Thr Ser 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 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 Ser Thr Thr Thr Thr Thr

165 170 175

Thr Ser Gly Ala Thr Ser Gln Ala Ala Ser Gln Ser Ala Ser 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 Thr Thr Ser Gly Ala Thr

340 345 350

Ser Gln Ala Ala Ser Gln Ser Ala Ser Ser Ser Ser Tyr Ser 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 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 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.

Images