WO2019242047A1

WO2019242047A1 - Recombinant spider silk proteins, preparation method therefor and industrial application thereof

Info

Publication number: WO2019242047A1
Application number: PCT/CN2018/094616
Authority: WO
Inventors: 马永; 赵百学; 王安良; 孙文秀; 江辰阳
Original assignee: 常州京森生物医药研究所有限公司
Priority date: 2018-06-22
Filing date: 2018-07-05
Publication date: 2019-12-26
Also published as: CN110627889A; CN110627889B

Abstract

Disclosed by the present invention are a recombinant spider silk protein series, coding genes thereof, expression and purification methods therefor, and an application of the recombinant spider silk protein series in biological materials such as sutures, bioremediation films and hemostatic materials. The recombinant spider silk protein series described in the present invention is composed of an N-terminal non-repeat region (N-NRT), a core repeat region (REP), and a C-terminal non-repeat region (C-NRT), wherein the core repeat region is composed of 1-30 RepAs and/or 1-15 RepBs, a single RepA amino acid sequence as shown in SEQ ID NO: 1, and a single RepB amino acid sequence as shown in SEQ ID NO: 2. The series of novel spider silk proteins for prokaryotic system soluble expression as designed by the present invention has high fermentation yield and high protein purity, spider silk membranes and spider silk fibers prepared from the spider silk protein series by means of a simple static spinning or wet spinning process have a mechanical strength of 2.5-6 MPa and 40-280 MPa respectively, and exhibit good cell compatibility and hemostatic properties, thus having prospects for industrial application in the medical field.

Description

Recombinant spider silk protein, preparation method and industrial application thereof

Technical field

The invention relates to a series of recombinant spider silk proteins, coding genes, expression and purification methods thereof, and the application of the recombinant spider silk proteins to medical products such as silk formation and nano-film formation.

Background technique

Spider silk is as hard as steel and as elastic as rubber. Its outstanding performance is mainly manifested in: high strength, high elasticity, high work of fracture, can be said to be the toughest material so far, known as "biosteel". With the deepening of research on spider silk, it was found that spider silk also has characteristics such as biodegradability, super shrinkage, high temperature resistance, low temperature resistance, and compatibility with biological tissues. Because of its unique physical and biological properties, spider silk has a wide range of applications in medicine, materials, military and textiles. Swedish scientist Jan Johansson uses non-irritating chemicals to produce artificial spider silk with good biocompatibility. It can be used in spinal cord repair or helping stem cell growth to repair damaged heart tissue and other regenerative medicine research. It can also be used for self-protection. Appliances and other textile industry applications (Nature, Chemical, Biology, DOI: 10.1038 / nchembio. 2269).

In view of the huge potential applicability of spider silk protein, scholars at home and abroad have strengthened their research on spider silk, hoping that spider silk can be used in practice on a large scale like silk. Because spiders cannot be domesticated and natural spider silk has little output, genetic engineering can be used to obtain spider silk in large quantities to meet the potential application requirements of spider silk. At present, researchers can use the expression systems of E. coli, yeast, insect cells, mammalian cells and other expression systems for the bioengineering of spider silk proteins. Among them, E. coli expression lines have multiple advantages such as fast growth, high yield, large scale production, low cost, simple culture conditions, and clear genetic background. They are currently widely used in the recombinant expression of spider silk proteins. However, the molecular weight of natural spider silk is very large (> 300KDa), and the expression of Escherichia coli is greater than 60KDa, and the expression will decrease. The natural molecular weight of natural spider silk is too large to be successfully expressed. For example, Fahnestock et al. Reported that spider traction silk protein was expressed in E. coli, but found that the expression efficiency of genes larger than 3 kb was reduced, and there was a phenomenon of gene abridged expression.

Studies have shown that spider silk proteins have four universal amino acid modules: (1) GPGXX; (2) GGx; (3) An / (GA) n; and (4) spacer. GPGXX (mainly two forms of GPGGx and GPGQQ) is a pentameric peptide, which mainly exists in traction silk proteins and flagellar silk proteins, forming a β-turn structure, and a series of β-turns are folded in series, similar to a spring structure. And this "spring" structure is related to the elasticity of spider silk protein, and the elasticity of spider silk protein is positively related to the content of the module. GGX forms a 3 ₁₀ -helix structure, that is, every three amino acids make up a helix. An / (GA) n forms a β-sheet structure. The molecular action between the structural sheets determines the strength of the spider silk protein, which is also one of the reasons that causes the spider silk to be insoluble in water. The spacer is a conserved region that separates glycine-rich regions. In general: (1) the chemical nature of spider silk is protein; (2) the amino acid sequence of spider silk protein is highly repetitive; (3) the amino acids in the spider silk protein are mainly alanine, glycine and serine; (4) It is rich in polyalanine and forms a β-sheet rigid structure; (5) β-ammonia corner structure formed by proline provides good elasticity for spider silk.

Researchers can design recombinant spider silk proteins based on the characteristics of the spider silk protein. The primary structure of the recombinant spider silk protein contains the above-mentioned modules that determine the mechanical properties of the spider silk protein. The sequence design can be personalized based on the research and development of different materials. The artificial spider silk protein obtained through artificial sequence design not only has controllable mechanical properties, but also can control the molecular weight of the protein in order to achieve efficient expression of the protein in the E. coli expression system.

The applicant found that based on the existing spider silk protein design scheme and preparation process, although it can meet the needs of general industrial applications, in the fields of medical products, not only the conventional tensile strength, but also high purity, Application requirements such as low immunogenicity cannot be met.

Summary of the Invention

In order to overcome the above technical problems, the inventors of the present application redesigned a series of protein sequences derived from natural spider silk, and simultaneously introduced a corresponding series of genes encoding recombinant spider silk protein sequences into host cells for protein expression, and established high efficiency Recombinant spider silk protein expression and preparation process. Based on this, the application of this series of recombinant spider silk proteins in electrospinning and wet spinning was studied.

The first object of the present invention is to provide a recombinant spider silk protein, which is composed of an N-terminal non-repeating region (N-NRT), a core repeating region (REP), and a C-terminal non-repeating region (C-NRT), wherein the core repeating region consists of 1 It consists of -30 RepA and / or 1-15 RepB. A single RepA amino acid sequence is shown in SEQ ID NO: 1, and a single RepB amino acid sequence is shown in SEQ ID NO: 2.

RepA and RepB can use the tandem itself or the hybrid tandem of the two as the core repeat region. When the self-tandem is used as the core repeat region, it is preferable to contain 5-30 RepA as the core repeat region or 5-15 RepB as the core repeat region.

When the hybrid tandem is used as the core repeat, a tandem of 1-5 RepA and 1-5 RepB is preferably used as the core repeat. More preferably, the tandem of 1-5 RepA is located near the N-terminal position of the entire fusion protein, and the tandem of 1-5 RepB is located at the C-terminal position of the entire fusion protein.

The amino acid sequence of the N-terminal non-repeating region (N-NRT) is shown in SEQ ID NO: 3, and the amino acid sequence of the C-terminal non-repeating region (C-NRT) is shown in SEQ ID NO: 4.

In the present application, the inventors performed gene synthesis in the form of five tandems (RepA5), and also performed gene synthesis in the form of five tandems (RepB5). In addition, in order to facilitate subsequent gene manipulation, the 5 ′ and 3 ′ ends of the nucleotides encoding RepA5 and RepB5 were introduced with homologous enzyme digestion sites BamHI and BglII, respectively.

The partial sequence involved in the present invention is shown in Table 1:

Table 1

The nucleotide sequences are codon optimized for the E. coli expression system, thereby increasing the expression in E. coli. The tandem nucleotide sequence can be prepared by PCR or by gene synthesis.

The present invention also provides an expression vector containing the above-mentioned nucleotide sequence, as long as the vector matches the E. coli expression system, preferably a vector with a high copy number and high expression efficiency, such as pET21b, pET28a, pBV220 and the like.

The invention also provides an E. coli strain, preferably BL21 (DE3), BL21 (DE3) plys, Rosetta (DE3), Transetta (DE3), etc., comprising the expression vector described above.

The invention also provides a method for producing high-density fermentation of recombinant spider silk protein, which specifically includes: activation of fermented seeds, preparation of fermented first-stage seed liquid, preparation of fermented second-stage seeds, and high-density fermentation. The high-density fermentation specifically includes the following steps:

Inserting the prepared secondary seed solution into a sterilized fermentation tank containing a batch fermentation medium according to an inoculation amount of 5-15%;

Set the fermentation temperature to 37 ° C, pH to 6.8-7.2, and DO to 30-40%;

After the start of fermentation, samples were taken periodically to determine the OD ₆₀₀ and the wet weight of the bacteria. When the DO curve showed a sharp rise, it indicated that the glucose in the batch medium was depleted and the feed culture was started (the flow acceleration of the feed medium was maintained at 8). -12g / L / h);

After the bacteria grow to between OD ₆₀₀ and 40-60, the fermentation temperature reaches 20-37 ° C. After the temperature is stabilized, add IPTG with a final concentration of 0.2-1.0mM to the fermentation tank to induce expression; induce expression 8-12h End cultivation.

The components of the batch fermentation medium include: 0.5-3 g / L of citric acid monohydrate, 8-15 g / L of potassium dihydrogen phosphate, 3-7 g / L of diammonium hydrogen phosphate, 10-20 g / L of glucose or 15 containing glycerin -30g / L, magnesium sulfate heptahydrate 1-3g / L. Before fermentation inoculation, a 1/1000 (V / V) trace element mother liquor is added to the batch fermentation medium to maintain the normal growth and metabolism of the bacteria.

The main components of the feed medium include: 1024g / L of glycerin, 10-20g / L of magnesium sulfate heptahydrate, and 30-60g / L of yeast powder. Before the start of feeding, 1/1000 must be added to the feed medium. (V / V) trace element mother liquor is used to maintain normal growth and metabolism of bacteria.

The components of the trace element mother liquid include: FeSO ₄ .7H ₂ O 10g / L, ZnSO ₄ .7H ₂ O 2.25g / L, CuSO ₄ .5H ₂ O 15g / L, MnSO ₄ .5H ₂ O 5g / L, CaCl ₂ .7H ₂ O 1g / L, CoCl.6H ₂ O 1g / L, Na ₂ MoO ₄ .2H ₂ O 1.125g / L, H ₃ BO ₃ 0.0625g / L, HCl 41.75ml, Biotin 0.5g / L.

The method for activating the fermented seeds is specifically: inoculating the constructed recombinant spider silk protein E. coli strain cryopreservation tube into a LB solid medium using a three-zone streaking method, and incubating at 37 ° C overnight for activation.

The method for preparing the first-stage fermented seed liquid is specifically: picking a single colony with a full shape and a moderate size from a solid medium and inoculating it into the LB liquid medium, and culturing at 37 ° C and 220 rpm for 8-10 hours in a shaker. Seed liquid.

The method for preparing the fermented secondary seeds is specifically: transferring the primary seed liquid into a fresh LB liquid medium according to an inoculation amount of 1%, and culturing at 37 ° C and 220 rpm in a shaker to between OD ₆₀₀ ≈3-5, This is a secondary seed liquid preparation.

Any of the above vessels and culture media used for bacterial culture should be filtered or humid-heat sterilized before use, and any culture medium should be added with kana mold at a final concentration of 50 μg / ml before use after cooling after sterilization. To ensure thoroughbred culture.

The invention also provides a method for purifying recombinant spider silk protein, including the following steps:

1. Take a certain amount of high-density fermentation bacteria and resuspend them to 100 g / L using buffer A (50 mM Tris, 100 mM sodium chloride, 1 mM EDTA, pH 8.0), and use the high-pressure homogenizer to perform the bacterial cells. broken.

2. Centrifuge the above bacterial lysate using a low-temperature centrifuge at 10,000 g for 30 minutes, discard the precipitate, and collect the supernatant.

3. Collect the supernatant of the bacterial lysate collected in step 2 by centrifugation, and put it in a water bath preheated to 75 ° C for 10-50min. The process of incubation should be continuously and gently stirred. This process is called Heat treatment of cell lysate.

4. Centrifugally collect the bacterial lysate after the heat treatment in step 3 to remove the precipitate, collect the supernatant, and filter the supernatant using a 0.8 μm aqueous membrane to remove particulate impurities.

5. Purification by IMAC affinity chromatography can obtain the target protein.

The inventors of the present application optimized a series of new spider silk protein sequences based on the reported amino acid sequences of spider silk proteins and the relationship between the structural characteristics and function of the spider silk proteins. Features in one. In addition, these sequences were respectively introduced into E. coli by genetic engineering technology and the corresponding high-density fermentation process was established to successfully achieve efficient prokaryotic soluble expression of spider silk proteins. In the past, in order to achieve the industrial application of spider silk protein, the designed spider silk protein is often designed to consider the amount of preparation under the premise of ensuring performance. However, for specific areas that require pure products such as medical products, the past design Spider silk protein has innate shortcomings, and even if it can obtain pure products, it needs to pay a huge investment. In view of this, the inventors have designed a series of spider silk proteins that can be expressed soluble in the prokaryotic system according to the properties of the spider silk protein. After purification, the purity of the recombinant spider silk protein (electrophoretic purity) can reach about 95%. The silk protein series of arachnoid membrane and arachnid fiber prepared through the static spinning or wet spinning process can have a mechanical strength of 2.5-6MPa and 40-280MPa, and have good cell compatibility and hemostatic effect, which can be fully satisfied. The special requirements in the medical field have the value of further development.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Construction principle of recombinant spider silk protein E. coli strain

Figure 2 Electrophoresis diagrams of recombinant spider silk proteins after purification in different designs

Figure 2-1 Electrophoretic detection of RepA5 purified sample, where M: protein molecular weight standard; lane1: RepA5 purified sample electrophoresis;

Figure 2-2 Electrophoretic detection of RepA10 purified samples, where M: protein molecular weight standard; lane1-2: electrophoresis of RepA10 purified samples;

Figure 2-3 Electrophoretic detection of RepA20 purified sample, where M: protein molecular weight standard; lane1: RepA20 purified sample electrophoresis;

Figure 2-4 RepB5 purified sample electrophoresis detection, where M: protein molecular weight standard; lane1: RepB5 purified sample electrophoresis;

Figure 2-5 Electrophoretic detection of RepB10 purified sample, where M: protein molecular weight standard; lane1: RepB10 purified sample electrophoresis;

Figure 2-6 Electrophoretic detection of RepB15 purified sample, where M: protein molecular weight standard; lane1: RepB15 purified sample electrophoresis;

Figure 2-7 Electrophoretic detection of RepA5RepB5 purified sample, where M: protein molecular weight standard; lane1: RepA5RepB5 purified sample electrophoresis;

Figure 2-8 Electrophoresis detection of RepB5 metal ion chelate chromatography purified sample, where M: protein molecular weight standard; lane1: before chromatography purification; lane2: chromatography purification penetration; lane3-4: chromatography purification elution sample;

Figure 3 Scanning electron microscopy image of the electrospinning sample of recombinant spider silk protein, where the first: RepA5, the second: RepA10; the third: RepA20; the first: RepB5; the second: RepB10; the third: RepB15; the next: RepA5RepB5; Next two: RepA5RepB5-RepA5RepB5

Figure 4 Mechanical test results of the electrospun fiber membrane of the recombinant spider silk protein, wherein the samples of the recombinant spider silk protein corresponding to Figure 4-1 to Figure 4-8 in this order are: RepB5, RepB10, RepB15, RepA5, RepA10, RepA20, RepA5RepB5, RepA5RepB5 -RepA5RepB5

Figure 5 Mechanical properties of the wet-spun fiber of recombinant spider silk protein. Figures 5-1 to 5-6 show the results of the mechanical properties of the RepB5, RepB10, RepB15, RepA5, RepA10, and RepA20 wet-spun fibers.

Fig. 6 Electron microscopic observation of the wet-spun fiber of recombinant spider silk protein. Fig. 6-1 is an electron microscopy of the RepA5 wet-spinning. Fig. 6-2 is a cross-sectional observation of the RepA5 fiber.

Fig. 7 Attachment effect of mouse fibroblasts (L929) on recombinant spider silk protein electrospun fiber membrane and bovine Achilles tendon collagen (type I) membrane, where 7-1 is collagen membrane, 7-2 RepB5 protein membrane;

Figure 8-1 Proliferation of mouse fibroblasts (L929) on two types of electrospun fiber membranes

Figure 8-2 Proliferation detection of human umbilical cord blood mesenchymal stem cells (MSC) on two types of electrospun fiber membranes

Fig. 9-1 Skin repair test of RepB5 and bovine Achilles tendon collagen electrospun fiber membranes, animal wound photos on day 1 and 22

Figure 9-2 Skin repair test results of RepB5 and bovine Achilles tendon collagen electrospun fiber membrane

Figure 10 RepB5 and bovine Achilles tendon collagen in vitro coagulation test results

detailed description

The following examples are further descriptions of this patent. It should be noted that the content described in the examples is only used to explain this patent and does not constitute a limitation on the invention content of this patent.

Example 1 Construction of recombinant spider silk protein E. coli expression strain

The invention relates to the construction of multiple recombinant spider silk protein strains. The splicing of repeat modules (repeat modules refer to RepB5 and RepA5) is achieved by molecular manipulation of isotail enzymes. The scheme flow is shown in Figure 1. The following describes the hybrid tandem (RepA5RepB5) formed by the tandem of RepA5 and RepB5 as an example, as follows:

1. Refer to the already published Argiopetrifasciata MaSp2 mRNA protein sequence in GenBank (GenBank gene accession number: AH015065.2), cut out the core repeat region from it to obtain the RepA and RepB nucleotide sequences, and form them after artificial assembly and codon optimization. The nucleotide sequences of RepA5 and RepB5 are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. In order to facilitate the subsequent molecular manipulation, without changing the amino acid sequence of RepA5 and RepB5, the restriction sites BamHI and BglII were introduced at the 5 'and 3' ends of the synthetic RepA5 and RepB5 artificial nucleotide sequences. RepA5 and RepB5 artificial nucleotide sequences were cloned into pUC57 plasmid, respectively, to form recombinant plasmids pUC57-RepA5 and pUC57-RepB5.

2. Synthesize the N-NRT and C-NRT gene sequences (such as SEQ ID NO: 16 and SEQ ID NO: 17), where the 5 'end of N-NRT contains the XbaI digestion site and the 3' end Contains BamHI digestion site (this digestion site is part of the N-NRT recombination nucleotide, does not change the amino acid sequence of N-NRT); C-NRT 5 'end contains BglII digestion site (the digestion As a part of the N-NRT recombination nucleotide, it does not change the amino acid sequence of N-NRT), the 3 'end contains XhoI digestion site; the nucleotide corresponding to N-NRT and C-NRT is synthesized in a fusion form , Which is N-NRT-C-NRT (5 '→ 3'). N-NRT-C-NRT was inserted into pET28aplus vector (recombined by removing the original BglII from pET28a original vector) by enzyme digestion and enzymatic ligation, and recorded as pET28aplus-N-NRT-C-NRT.

4. pUC57-RepA5 and pUC57-RepB5 were digested with BamHI and BglII to obtain RepA5 and RepB5 fragments with corresponding sticky ends. PET28aplus-N-NRT-C-NRT was digested with BamHI and BglII to obtain a vector backbone fragment with corresponding sticky ends. The viscous fragment obtained by enzymatic digestion was ligated and transformed into E. coli Transetta (DE3) to form a recombinant expression strain.

5. Pick a single clone from the transformation plate in step 4 for culture, extract the plasmid and use BamHI and BglII to digest and identify, and analyze and identify the positive clones obtained according to the principle of cleavage of the same tail. The positive clones are Transetta (DE3). ) -RepA5 and Transetta (DE3) -RepB5. The corresponding positive plasmids are pET28aplus-N-NRT-RepA5-C-NRT and pET28aplus-N-NRT-RepB5-C-NRT.

6. pET28aplus-N-NRT-RepA5-C-NRT was digested with BglII to obtain a vector backbone fragment with corresponding sticky ends, and pUC57-RepA5 was digested with BamHI and BglII to obtain a RepA5 fragment with corresponding sticky ends. The two cohesive fragments were ligated and transformed into E. coli Transetta (DE3) to form a recombinant expression strain. After identification, Transetta (DE3) -RepA10 (a tandem containing 10 RepA) was obtained. The corresponding positive plasmid was pET28aplus-N-NRT. -RepA10-C-NRT. Similarly, pET28aplus-N-NRT-RepA10-C-NRT can be repeatedly operated to obtain a tandem containing n RepA5 (such as a tandem containing 5, 10, 20, 30 ... RepA) recombinant E. coli strain, That is Transetta (DE3) -n × RepA5.

7. In the same way, transpose pUC57-RepB5 and pET28aplus-N-NRT-RepB5-C-NRT according to steps 4-6, respectively, to obtain Transetta (DE3) -n × RepB5 (a tandem body containing n RepB5 ).

8. Digest pET28aplus-N-NRT-RepA5-C-NRT plasmid with BglII to obtain the vector backbone fragment with the corresponding sticky ends, pET28aplus-N-NRT-RepB5-C-NRT to digest the band with BamHI and BglII RepB5 fragment with corresponding sticky ends. RepB5 with a sticky end was linked to pET28aplus-N-NRT-RepA5-C-NRT with a sticky end and transformed into E. coli Transetta (DE3) to form a recombinant expression strain. A single clone was selected from the transformation plate and cultured. The plasmid was extracted and identified by digestion with BamHI and BglII. The positive clone obtained was analyzed and identified according to the principle of cleavage of the same tail enzyme. The positive clone was Transetta (DE3) -RepA5RepB5. The positive plasmid was pET28aplus-N-NRT-RepA5-RepB5-C-NRT.

9. Sequencing the strains identified as positive by the above enzyme digestion to verify whether the sequences are correct.

Example 2 High-density fermentation of recombinant spider silk protein E. coli strains

Spider silk protein contains a large number of repetitive amino acid sequences. This characteristic causes it to encounter problems such as depletion of the tRNA pool, obstacles to gene replication, truncation of protein translation, etc., resulting in low expression levels. Studies have shown that as the molecular weight of artificial spider silk protein increases (greater than 60KDa), the expression of the protein will increase greatly, and the problem of expression failure may occur. The molecular weight of natural spider silk protein can reach more than 200KDa. Although there is no direct research showing that the molecular weight of spider silk protein is directly proportional to its mechanical properties, it is still necessary to make the artificially designed spider silk protein as close as possible to the natural spider silk protein in molecular weight. similar. Therefore, the recombinant spider silk protein must balance molecular weight with the expression of E. coli. If the molecular weight is too large, the expression will be reduced or the expression will fail. If the molecular weight is too small, the biomimetic degree of the natural sequence will be low.

This section describes the inventor's high-density fermentation process for recombinant spider silk protein, which is illustrated by the fermentation process of Transetta (DE3) -RepA20 (The molecular weight of RepA20 in the spider silk protein construct in Example 1 is relatively large, reaching 88 KDa The evaluation of process yield is representative.

1. Inoculate the constructed recombinant recombinant spider silk protein E. coli strain cryopreservation tube into the LB solid medium using a three-zone streaking method, and incubate at 37 ° C, 220 rpm with a shaker overnight for activation;

2. Pick a single colony with a full shape and a moderate size from the solid medium and inoculate it into the LB liquid medium. Incubate at 37 ° C and 220 rpm for 8-10 hours. This is the first-level seed solution.

3. Transfer the primary seed solution to fresh LB liquid medium at 1% inoculum, and culture at 37 ° C and 220 rpm in a shaker until OD ₆₀₀ ≈3-5. This is the secondary seed solution preparation.

4. The prepared secondary seed liquid is inoculated into the fermentation medium to start feeding-batch high-density fermentation culture. The high-density fermentation process can realize the large-scale expression of recombinant spider silk protein. The components of the batch fermentation medium include: 1.7 g / L of citric acid monohydrate, 12 g / L of potassium dihydrogen phosphate, 4 g / L of diammonium hydrogen phosphate, 30 g / L of glycerin, 1.2 g / L of magnesium sulfate heptahydrate, Before fermentation inoculation, a 1/1000 (V / V) trace element mother liquor is added to the batch fermentation medium to maintain the normal growth and metabolism of the bacteria. The trace element mother liquor components include: FeSO ₄ .7H ₂ O 10g / L, ZnSO ₄ .7H ₂ O 2.25g / L, CuSO ₄ .5H ₂ O 15g / L, MnSO ₄ .5H ₂ O 5g / L, CaCl ₂ .7H ₂ O 1g / L, CoCl.6H ₂ O 1g / L, Na ₂ MoO ₄ .2H ₂ O 1.125 g / L, H ₃ BO ₃ 0.0625 g / L, HCl 41.75 ml, Biotin 0.5 g / L. The main components of the feed medium include: 1024 g / L of glycerin, 20 g / L of magnesium sulfate heptahydrate, and 50 g / L of yeast powder. Before the start of the feeding, the above-mentioned trace element mother liquor of 1/1000 (V / V) is added to the feeding medium to maintain the normal growth and metabolism of the bacteria. The steps of the high-density fermentation method include: inserting the prepared secondary seed liquid into a sterilized fermentation tank according to an inoculation amount of 5-15% → setting the fermentation temperature to 37 ° C and the pH to 6.8-7.0 The DO is set between 30-40% (controlled by the speed / air / high-purity oxygen correlation) → regular sampling after the start of fermentation to measure OD ₆₀₀ and the wet weight of the bacteria, until the DO curve shows a sharp rise It indicates that the glucose in the batch medium is depleted, and the feed culture is started (the flow acceleration of the feed medium is maintained at 12 g / L / h (feed medium quality / fermentation liquid initial volume / time)) → wait for bacteria When the body grows to between 0D ₆₀₀ ≈ 40-55, reduce the fermentation temperature to 30 ° C. After the temperature is stabilized, add IPTG with a final concentration of 0.5 to the fermentor to induce expression → induce the expression and end the culture 6h.

Any of the above vessels and culture media used for bacterial culture should be filtered or humid-heat sterilized before use, and any culture medium should be added with kana mold at a final concentration of 50 μg / ml before use after cooling after sterilization. To ensure thoroughbred culture. The recombinant spider silk protein contains a large number of amino acid repeats. The strain obtained in Example 1 can achieve effective expression using this high-density fermentation process, and some strains can reach more than 0.5 g / L, which lays the foundation for the large-scale application of recombinant spider silk.

Example 3 Purification and Preparation of Recombinant Spider Silk Protein

This section describes the non-chromatographic protein purification method of spider silk protein. After simple purification, the purity of the recombinant spider silk protein (electrophoretic purity) can reach more than 75%. It needs to be clear that its residual impurities are important for subsequent Spinning and other operations have no obvious impact, and the pure spider silk protein can meet most industrial applications. Because the artificial spider silk protein in the present invention contains a 6 × His tag at the N-terminus, and the designed molecule is soluble, the artificial spider silk protein in this patent can be more purified by IMAC affinity chromatography. The electrophoretic purity is above 95%, which meets the requirements of refining and purification, and can completely meet the needs of special applications such as medical treatment. In this embodiment, the fermented bacterial cells prepared in Example 2 are respectively processed by simple purification and refining purification processes, and the specific purification operations are as follows:

3. Collect the supernatant of the bacterial lysate collected in step 2 by centrifugation, and place it in a water bath preheated to 75 ° C for 20 minutes. During the heat preservation process, continuous gentle stirring is required. This process is called bacterial cells. Thermal treatment of lysate.

5. Add the supernatant after the filtration treatment in step 4 to a 3M aqueous ammonium sulfate solution for salting out. Add ammonium sulfate slowly, and the salting-out system is in a state of rapid stirring and mixing. With the addition of ammonium sulfate, when the salting-out system appears turbid, stop adding ammonium sulfate, and place the salting-out system at 4 ° C with stirring for 5 h.

6. Centrifuge the salting-out system in step 5 to collect the salting-out precipitate. The precipitate contains artificial spider silk protein.

7. The artificial spider silk protein salted-out precipitate in step 6 is repeatedly washed with a 20-50% ethanol aqueous solution until the ammonium sulfate is substantially removed (the conductivity of the washing solution is not higher than 0.05 mS / cm), and then collected The spider silk protein is precipitated, and the spider silk protein raw material is obtained after lyophilizing the protein.

A part of the precipitated protein was taken for SDS-PAGE analysis using the method in Example 1. The analysis result is shown in FIG. 2. The electrophoresis detection results were obtained by using gray scale analysis of various artificial spider silk proteins to obtain electrophoretic purity of more than 75%. The final protein collected by centrifugation can be dried by freeze drying, hot air drying, spray drying and other methods to obtain purified spider silk protein. After drying, the spider silk protein is weighed, and the recombinant spider silk protein is calculated based on the purity and fermentation volume. The fermentation yield of RepA20 was 0.45 g / L.

At the same time, the inventor also selected RepB5 for the purification research of IMAC affinity chromatography. The affinity chromatography uses the sample heated in step 4 of this example as the initial sample to be purified (EDTA should be removed from the sample). Purification method Refer to Gehealthcare's purification scheme for nickel ion chelate chromatography. The electrophoretic analysis of the purified sample is shown in Figure 2-8. According to the analysis, the target protein can be effectively combined with the chromatographic packing, and the electrophoretic purity can be more than 95% after purification, which meets the requirements of purification.

The applicant used the fermentation and purification processes of Example 2 and Example 3 to ferment and purify the partially constructed recombinant spider silk protein E. coli strain, respectively. Based on the results calculation, the protein expression levels of different recombinant spider silk protein strains are shown in Table 2 As shown.

Table 2

Example 4 Preparation of Electrospinning Dope of Recombinant Spider Silk Protein and Electrospinning

1. Take the dried recombinant spider silk protein sample and use HFIP (hexafluoroisopropanol) to dissolve it. The feeding amount is 10% (wt%). Stir and dissolve at room temperature for more than 48h until the solution is uniform and transparent.

2. Take 2ml of the spinning dope and add it to the syringe. Select the 27G needle as the injection needle, set the injection rate to 1ml / h, set the voltage to 16KV, set the receiving distance to 15cm, and the width of the spinning head to the left and right. 8-12cm, the receiving substrate is tin foil, and the fiber membrane is prepared by electrostatic spinning.

3. The fiber membrane prepared by the above-mentioned electrospinning was detected and analyzed using a scanning electron microscope, and the analysis result is shown in FIG. 3.

4. The fiber membrane prepared in step 2 is cut with a cutter (to remove the edge part of the spinning radiation) into a strip shape with a width of 1 cm.

5. Peel the fiber film prepared in step 4 from the foil, use a digital spiral micrometer to measure the thickness of the fiber film, and then use a Shimadzu universal tensile tester to perform a tensile test (using a 50N sensor with a test speed of 10mm) / min, relative humidity is 40%, test distance is 2cm, preload is 0.1N), test results are shown in Figure 4. It can be seen from Figures 3 and 4 that the use of the spider silk protein of this patent can meet the requirements for the spinnability of the protein by electrospinning, and the fiber membrane is uniform and complete in the macro. Scanning electron microscopy shows a typical fiber morphology in the micro. It is shown that the mechanical strength of the recombinant spider silk protein fiber membrane prepared by electrostatic spinning is between 2.5-6MPa.

6. At present, artificial skin that can be used for medical treatment requires that the strength of the material should be between 2-17 MPa. Therefore, the fiber membrane obtained by the electrospinning has mechanical application strength of skin-like medical materials.

Example 5 Preparation of wet spinning solution of recombinant spider silk protein and wet spinning

1. Take the dried protein sample and dissolve it with HFIP (hexafluoroisopropanol) solvent. The protein concentration is 20% (w / v%). Stir and dissolve at room temperature for more than 10h until the solution is uniform and transparent.

2. Remove the foam from the spinning solution in step 1, and then inject it into a 1ml syringe. Use a 27G injection needle to push it out of the syringe. The spinning solution is squeezed out of the needle and dipped in ethanol to be solidified to form fiber filaments.

3. Take out the solidified fiber filament in step 2 and stretch it for 3-5 times in 80 ° C steam, and then expose it to 150 ° C for 30s to obtain mature fiber filament.

4. Use the Shimadzu universal tensile tester for tensile testing of the mature fiber yarn in step 3 (using a 50N sensor, a test speed of 10mm / min, a relative humidity of 40%, a test distance of 2cm, and a preload of 0.1N) The test results are shown in Figure 5. The mechanical strength is between 220 and 280Mpa.

5. Observe the surface and section of the fiber under the scanning electron microscope with the mature fiber in step 3 and the fiber broken in the mechanical measurement in step 4, and the results are shown in FIG. 6.

6. The fiber filament prepared by the method has a relatively regular cylindrical shape, the surface is smooth with obvious grooves, and the tensile fracture surface is relatively dense. Compared with the mechanical strength (40-280 MPa) of artificial recombinant spider silk fiber filaments reported at present, the mechanical strength of the fiber filaments corresponding to several recombinant spider silk proteins designed by the inventors belongs to a higher level.

Example 6 Compatibility Evaluation of Electrospinning Membrane of Recombinant Spider Silk Protein

The recombinant spider silk protein fiber membrane prepared by the inventor's electrospinning method has the characteristics of an electrospun fiber membrane in physical appearance. Therefore, in order to evaluate the biological compatibility of the fiber membrane, we used human umbilical cord blood mesenchyme Adhesion and appreciation of MSCs and mouse fibroblasts (L929) were evaluated. At the same time, it is recognized in the industry that natural collagen has excellent biocompatibility, is beneficial to skin repair and coagulation, and is reflected in the characteristics of promoting cell attachment and value-added. Therefore, we used bovine Achilles tendon collagen in the experiments. Comparison of electrospun membranes. For the preparation of bovine Achilles tendon collagen electrostatic spinning membrane, refer to the method in Example 5, and based on this method, the choice of solvent for protein dissolution is adjusted. The thickness of the prepared two kinds of protein electrostatic spinning membranes is consistent. Specific cytocompatibility evaluation methods are as follows:

1. Take the freshly prepared bovine Achilles tendon collagen and the recombinant spider silk protein RepB5 primary membrane for vacuum spinning for 12h to remove the non-volatile clean solvent in the fiber membrane. The former is crosslinked and fixed by chemical crosslinking method, and the latter Physical fixation is used to reduce the water solubility of the two proteins.

2. The fiber membrane fixed in step 1 is cut into a suitable size according to the diameter of the cell culture dish hole to ensure that the fiber membrane can evenly and completely cover the bottom of the culture dish.

3. After soaking the round fiber membranes of the two proteins cut in step 2 with 75% ethanol for 2 hours, respectively, take them out and spread them on the bottom of a 24-well plate and dry them. Collagen membranes were spread on 2 plates, each with 6 wells, and recombinant spider silk protein RepB5 membranes were spread on 2 plates, each with 6 wells. After air-drying, UV irradiation was performed for 1 hour.

4.L929 cell plating

Aspirate the culture solution from the T75 cell culture flask. After washing it in PBS, add 1 mL of trypsin. Gently shake the culture flask to allow the trypsin to contact all the cells at the bottom of the cell culture flask. Then, aspirate the trypsin to culture the T75 cells. The flask was placed at 37 ° C. After 3 minutes of digestion, the culture flask was removed and the cells were separated from the bottom of the cell flask. 10 mL of 1640 medium containing 10% FBS was added to the culture flask. After gently pipetting, the 10 mL cell suspension was aspirated and added to a 50 mL centrifuge tube. A small amount of the cell suspension was placed in a 1.5 ml centrifuge tube. Use a blood cell count. Plates are counted. This was prepared as a cell suspension of 4 × 10 ⁴ cells / ml. In a 24-well plate, wells with and without membranes are plated, and 200 ul of cell suspension is added to each well followed by 300 μl of medium for culture.

5.MSC plating

Aspirate the culture solution from the T75 cell culture flask. After washing it in PBS, add 1 mL of trypsin. Gently shake the culture flask to allow the trypsin to contact all the cells at the bottom of the cell culture flask. Then, aspirate the trypsin to culture the T75 cells. The flask was placed at 37 ° C. After 3 minutes of digestion, the culture flask was removed and the cells were separated from the bottom of the cell flask. Add 10 mL of DMEM / F-12 medium containing 10% FBS to the culture flask. After gently pipetting, mix 10 mL of the cell suspension and add it to a 50 mL centrifuge tube. Take a small amount of the cell suspension into a 1.5 mL centrifuge tube. , Use a blood cell counting board for counting. This was prepared as a cell suspension of 4 × 10 ⁴ cells / ml. In a 24-well plate, wells with and without membranes are plated, and 200 ul of cell suspension is added to each well followed by 300 μl of medium for culture.

6.Detection of cells on bovine Achilles tendon collagen film and spider silk protein film

On the 3rd, 4th, and 5th days, one 24-well cell culture plate of L929 cells was taken out, observed and photographed with a microscope, and the results are shown in FIG. 7. As the MSC cells are attached to the fiber membrane, the light transmittance will decrease. Imaging was not possible under the microscope, so no photos were taken for observation. It can be seen from FIG. 7 that both the bovine Achilles tendon collagen film and the recombinant spider silk protein fiber film can effectively achieve the attachment of L929, and the cell shape is good. From the perspective of proliferation effect, for the recombinant spider silk protein fiber membrane, it showed significantly better value-added effect than the collagen fiber membrane in both L929 and MSC cell culture.

In the removed 24-well cell culture plate, aspirate all the medium from each well, then add 200ul of culture medium to each well, add 20ul of CCK-8 solution, and incubate at 37 ° C for 1h. Use a microplate reader at 450mm Readings, statistics of the results, and analysis of cell proliferation, the results are shown in Figures 8-1 and 8-2.

Therefore, it can be judged that, compared with the two kinds of fiber membranes, the recombinant spider silk protein fiber membrane can achieve cell attachment and growth faster, and therefore has good biocompatibility.

Example 7 Skin Regeneration Test of Recombinant Spider Silk Protein Electrospinning Film

Skin wound repair and regeneration is an important potential field of application for electrospun fiber membranes. The inventors evaluated recombinant spider silk protein fiber membranes prepared with electrospinning method together with natural collagen membranes in skin wound repair. Role, specific evaluation methods and results are as follows:

Animal model establishment: Using the method of self-control, the rats selected three circular areas with a diameter of 12 mm at equal distances at about 0.5 cm on both sides of the spine, and each point was separated by 1 cm. Rats were injected intraperitoneally with a 10% chloral hydrate solution at a dose of 3.0 mL / kg. After the normal reflection disappeared, the full-thickness skin tissue was peeled off from the circular area and covered with the test product RepB5 and the bovine Achilles tendon collagen fiber membrane. After the experiment, each patient was injected with 2.0 × 10 ³ U / d ampicillin sodium for 3 consecutive days to prevent infection. Wound healing was observed after surgery. Rats were euthanized at 3 weeks after operation. Digital images of the surgical site were collected and the area of the wound / hairless area of the surgical department was calculated using software to investigate the role of each group of samples in promoting wound healing. The results are shown in the figure below. 9 shown.

It can be seen from the skin wound repair and regeneration test that the skin repair effect of the recombinant spider silk protein electrospun fiber membrane is better than that of the bovine Achilles tendon collagen fiber membrane, and the wound recovery is better after surgery.

Example 8 In vitro coagulation test of recombinant spider silk protein

At present, the relatively high-end medical coagulation products on the market include powdered natural collagen, which has the characteristics of rapid coagulation and biosafety. Therefore, the inventors also investigated the in vitro coagulation effect of recombinant spider silk protein compared with natural collagen, as follows:

1. Raw material acquisition: Reconstituted spider silk protein lyophilized powder and bovine Achilles tendon collagen samples were crushed and powdered under the same conditions, and passed through a 50-mesh sieve to obtain a uniform powdery test product with substantially uniform particle size.

2. Put equal-quality bovine Achilles tendon collagen powder and RepB5 powder in the blood collection tube and evacuate them respectively; the blank control tube is evacuated directly, 3 tubes each.

3. Take 11 to 12 weeks of age, one ordinary New Zealand white rabbit, male or female, weighing 1.8 to 2.2 kg, rabbits were injected intraperitoneally with a 10% chloral hydrate solution at a dose of 3.5 mL / kg, and the positive reflection disappeared after turning 3ml blood was collected through the common carotid aorta to each blood vessel. After the blood was added to the vacuum tube, the timing was started. At room temperature, the test tube was tilted every 5 seconds, and when the blood no longer flowed, the timing was stopped to obtain the whole blood coagulation time. The calculation results are shown in Figure 10.

From the test tube coagulation test results, it can be seen that under the same test conditions, the protein powder prepared by the spider silk protein RepB5 has a shorter clotting time than the bovine Achilles tendon collagen powder, so it is judged that it has good procoagulant potential.

Claims

Recombinant spider silk protein, the amino acid sequence of which consists of N-terminal non-repeating region, core repeating region, and C-terminal non-repeating region, wherein the core repeating region is composed of 1-30 RepA and / or 1-15 RepB, and a single RepA amino acid sequence As shown in SEQ ID NO: 1, the single RepB amino acid sequence is shown in SEQ ID NO: 2; the N-terminal non-repeating region, the amino acid sequence is shown in SEQ ID NO: 3; the C-terminal non-repeating region, the amino acid The sequence is shown in SEQ ID NO: 4.
The recombinant spider silk protein according to claim 1, wherein the core repeat region consists of 5-30 RepA or 5-15 RepB.
The recombinant spider silk protein according to claim 1, wherein the core repeat region is composed of 1-5 RepA and 1-5 RepB tandems.
The recombinant spider silk protein according to claim 3, wherein the tandem of 1-5 RepA is located near the N-terminus of the entire fusion protein, and the tandem of 1-5 RepB is located in the entire fusion protein C-position.
The recombinant spider silk protein according to any one of claims 1 to 4, wherein the core repeat region is composed of 5 RepA tandems, 10 RepA tandems, 20 RepA tandems, and 30 RepA tandems. , 5 RepB tandems, 10 RepB tandems, 15 RepB tandems, 5 RepA plus 5 RepB tandems, or 2 RepA5RepB5 tandems.
A vector comprising the nucleotide sequence according to claim 5.
An E. coli strain comprising the vector of claim 6.
A method for expressing a recombinant spider silk protein, comprising the following steps:

(1) The E. coli colony according to claim 7 is inserted into a fermentation tank, and the fermentation temperature is set to 37 ° C, the pH is between 6.8-7.2, and the DO is set between 30-40%;

(2) After the start of fermentation, take regular samples to measure the OD600 and the wet weight of the bacteria. When the DO curve rises sharply, start feeding culture, and the flow acceleration of the feeding medium is maintained at 8-12g / L / h;

(3) When the bacterial cells grow to between OD600≈45-55, reduce the fermentation temperature to 25-30 ° C. After the temperature is stabilized, add IPTG with a final concentration of 0.2-1.0mM to the fermentation tank to induce expression; The culture was terminated at -12h.
A method for purifying recombinant spider silk protein, comprising the following steps:

(1) Take E. coli expressing recombinant spider silk protein, crush the cells, and collect the lysate supernatant;

(2) take the above lysate supernatant membrane and filter to remove impurities;

(3) The target protein can be obtained by purification by IMAC affinity chromatography.
The application of the recombinant spider silk protein according to any one of claims 1 to 5 in the production of protein sutures, protein bioremediation membranes, and protein hemostatic materials.