33 results about "Spidroin" patented technology

The invention belongs to the technical field of biology, and particularly discloses a high-density fermentation method and application of recombinant spider silk protein, and the high-density fermentation method comprises the following steps: firstly, transforming an expression vector carrying spider silk protein genes into modified pichia pastoris cells to obtain a strain; the activated strain is subjected to expanding culture, high-density fermentation culture and purification, and recombinant spider silk protein is obtained; wherein the transformation means of the pichia pastoris cell is to integrate Vitreoscilla hemoglobin Vhb gene into the pichia pastoris cell by utilizing a gene recombination technology, and the expression of the Vitreoscilla hemoglobin Vhb gene is improved by carrying out metabolic engineering transformation on the pichia pastoris cell for expression and optimizing the regulation and control of staged fermentation parameters, so that the expression efficiency of the Vitreoscilla hemoglobin Vhb gene is improved. The problems that in existing recombinant protein fermentation, oxygen dependence is high, product degradation is serious, purity is low, and large-scale stability is poor are solved, the yield and purity of recombinant spider silk protein are guaranteed, and large-scale production of the recombinant spider silk protein is facilitated.

The invention belongs to the technical field of protein, and aims to solve the problem that the application range of spider silk particles as a drug carrier is limited due to the fact that the spider silk particles are in negative net charge under a neutral condition and can only load drugs with positive charges or neutral charges at present. The novel recombinant spider silk protein is prepared on the basis of an N connection sequence of araneus ventricosus MaSp1, has positive net charge under a neutral pH value, can load a negatively charged drug, and expands the application range of the spider silk protein as a drug carrier; and a drug release test result shows that the recombinant spider silk protein is superior to MaSp2-based silk protein particles in the aspect of etoposide delivery.

The invention relates to the technical field of biology, in particular to a recombinant coral-spider silk fusion protein and functional alginate fiber and application thereof. The recombinant coral-spider silk fusion protein provided by the invention comprises the recombinant coral-spider silk fusion protein formed by fusing spider silk protein or rattan silk protein and coral protein. The invention designs a fusion protein only containing spider silk protein and coral protein. The recombinant coral-spider silk fusion protein is applied to alginate fibers, colored spinning of the alginate fibers can be achieved, and the spun fibers are high in color fastness, superior to original fibers in strength and elongation and bright and stable in color.

The invention belongs to the technical field of biology, particularly discloses a method for preparing spider silk protein-containing composite silk by using transgenic silkworms and application of the spider silk protein-containing composite silk, and particularly provides a method for synthesizing and secreting spider silk protein by using a silkworm silk gland bioreactor. The spider silk protein gene is introduced into a bombyx mori genome in a virus infection manner by utilizing a transgenic bombyx mori technology and is specifically expressed in bombyx mori silk gland cells, and the spider silk protein target gene can be introduced in batches by virtue of a lentivirus transfection method, so that bombyx mori capable of synthesizing and secreting complete spider silk protein can be developed on a large scale; the spider silk protein is synthesized and secreted through bombyx mori silk gland cells and enters silkworm cocoons along with the spinning and cocooning behaviors of bombyx mori, the silk is collected, the spider silk protein-containing composite silk with the bombyx mori silk as the background is obtained, a foundation is laid for mass production of black spider silk protein, and meanwhile a foundation is laid for improving the mechanical performance of the silk.

The invention relates to the technical field of biology, in particular to recombinant spider silk protein, modified polyurethane and application of the recombinant spider silk protein and the modified polyurethane. The recombinant spider silk protein provided by the invention comprises spider silk protein or spider silk-like protein; the spider silk protein or spider silk-like protein comprises at least one spider silk globulin NT, at least one spider silk protein repeat region and at least one spider silk globulin CT from the N end to the C end. Research finds that when the recombinant spider silk protein is applied to waterborne polyurethane, the mechanical strength of a polyurethane material is remarkably improved, the mechanical property of the polyurethane material is enhanced, and the polyurethane material is environmentally friendly and non-toxic, has the characteristics of no toxicity, high strength, high elasticity and the like, and keeps the original characteristics of low organic matter emission, no pollution, high elasticity, air permeability and the like at the same time. The recombinant spider silk protein can have a CBM structure, so that the mechanical property can be improved. Fluorescent protein and / or chromoprotein can be fused on the recombinant spider silk protein, polyurethane with different appearances can be obtained, and color combination is firm.

The invention provides a spider silk protein modified body easy to purify as well as a modification method and a purification method thereof, a plurality of amino acid residues in flexible regions on two sides of a spider silk protein microcrystalline region are replaced with acidic amino acids or alkaline amino acids, so that the proportion of charged amino acids in the flexible regions is greater than or equal to 7%, and the spider silk protein modified body is obtained; the spider silk protein modified body is purified in the form of an inclusion body through a temperature change process of heating and cooling. The modified body can be simply purified with high recovery rate through simple heating and cooling processes, the total recovery rate is up to 98% or above, the purity is 90%-99% or above, and meanwhile, the formed artificial spider thread is endowed with higher tensile strength by strengthening the interaction of beta-sheet on the two sides of a microcrystal region, especially between different chains. A foundation is further laid for large-scale efficient production and low-cost separation and purification of the high-performance spider silk protein, and the application possibility of the spider silk protein in various fields is greatly enhanced.

The present invention relates to a novel injectable protein delivery system and a method for delivering one or more therapeutic agents to the central nervous system (CNS) to promote axonal regeneration. The system is based on the use of a recombinant spider silk protein, referred to as spiroin-SpyTag, which undergoes a rapid transition from a sol state to a gel state when exposed to ultrasonic treatment and incubated at body temperature. This unique characteristic allows the material to be easily injected into a specific target tissue. The methods disclosed herein can deliver a protein therapeutic agent covalently conjugated to spiroin-SpyTag to a subject affected by a CNS disorder or injury. In addition, methods of making such injectable protein delivery systems are fast, convenient, and economical and effective.

The application provides an aggregated spider silk protein nanoparticle, a drug delivery system and a preparation method and application thereof, and belongs to the technical field of biological medicines.The application provides application of the aggregated spider silk protein in preparation of a drug delivery system.The application researches and finds that the aggregated spider silk protein nanoparticle prepared by using the amino acid sequence such as SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.5 has good colloidal stability and good safety, and is a novel drug delivery system.The drug delivery system loaded with a tumor drug has high encapsulation efficiency and drug loading capacity for the tumor drug, the encapsulation efficiency is more than 60%, has a slow-release effect on the tumor drug, and is an excellent drug delivery system.The research finds that compared with the simple tumor drug, the drug delivery system loaded with the tumor drug has a significantly improved killing effect on tumor cells, and can effectively treat tumors.

The present disclosure is directed to systems and methods for synthesizing a spidroin. In some embodiments, the methods comprise synthesizing a monomer in vivo in a heterologous host, the monomer comprising an N-terminus IntC domain and a C-terminus IntN domain, and post-translationally polymerizing the synthesized monomer via in vitro split-intein mediated polymerization.

The invention discloses a natural spider silk-based self-adaptive surgical suture and a preparation method thereof, and belongs to the technical field of biomedical materials. According to the surgical suture, natural spider silk serves as a raw material, protein chains in the spider silk are rearranged in order by regulating and controlling the moisture state, applying axial pre-stretching strain and conducting drying, annealing and shaping under the limited condition, and the suture material with the high strength and the environmental response characteristic is obtained. The surgical suture is based on natural spider silk, has excellent biocompatibility and inherent biodegradability, becomes an ideal candidate material for biomedical application, and is successfully applied to suture of skin, stomach and other parts. According to the invention, the spider silk protein chains are rearranged into the superfine nanofibers, so that the engineered natural spider silk suture with remarkably enhanced mechanical and functional properties can be produced, and the monofilament can realize the breaking strength of 2.0 GPa, the toughness of 480 MJ m < 3 > and the driving stress of 92.6 MPa.

Disclosed in the present application is a method for preparing a regenerated cellulose fiber incorporating a recombinant protein, the method comprising the following steps: dissolving a recombinant protein and cellulose in a solvent for uniform mixing, so as to obtain a spinning solution; and using the spinning solution to perform spinning, so as to obtain a regenerated cellulose fiber incorporating the recombinant protein, the recombinant protein being a recombinant protein obtained by fusion expression of at least one cellulose binding domain and at least one functional protein. In the present application, CBM-containing recombinant functional proteins can maintain structural and functional stability in NMMO systems, so as to provide the biological activity of proteins for Lyocell fiber systems; incorporation of fluorescent proteins can impart natural fluorescence to fibers, which have application prospects in the field of textiles and fashion; expression of MaSp1 recombinant spider silk proteins enhances the fiber strength, and expression of MaSp4 recombinant spider silk proteins improves the elastic properties of fibers; and in addition, CBM fusion proteins can also carry other functional modules, so as to endow cellulose fibers with specific functionalities.

The application discloses a kind of recombinant spider silk protein fibers based on SpiCE-DS8 protein and preparation method thereof, belong to polymer fiber material technical field.The application discloses the key functional domain SpiCE-DS8-S (KGGLDILGGLLKLSKAGKDGKNFF) of SpiCE-DS8 protein, which can promote the self-assembly and fiber formation of silk protein.By SpiCE-DS8 protein as the raw material of spinning solution, the recombinant spider silk protein fiber prepared has significant improvement in strength, toughness and elongation, which provides the possibility for improving the mechanical properties of in vitro recombinant silk, and the method of the application simplifies the spinning process, continuous spinning can be realized, which is beneficial to industrial production.

The subject invention pertains to a novel injectable protein delivery system and methods for delivering one or more therapeutic agents in the central nervous system (CNS) for promoting axon regeneration. This system is based on the use of a recombinant spider silk protein called spidroin-SpyTag, which undergoes a rapid transition from a sol state to a gel state when exposed to ultrasound treatment and incubated at body temperature. This unique characteristic allows the easy injection of the material into a specific target tissue. The methods herein disclosed allow the delivery of protein therapeutics covalently conjugated to the spidroin-SpyTag to a subject affected by a CNS disorder of injury. Additionally, the method for fabricating this injectable protein delivery system is rapid, convenient, and cost-efficient.

A recombinant spider silk protein comprises an N-terminal (NT) domain, a repetitive region (REP) domain and a C-terminal (CT) domain. The REP domain comprises at least 100 amino acids derived from the repetitive region of a spider silk protein of Larinioides sclopetarius. The NT domain is derived from the NT domain of a spider silk protein other than the spider silk protein, from which the REP domain is derived and / or the CT domain is derived from the CT domain of a spider silk protein other than the spider silk protein from which the REP domain is derived.

The invention relates to the technical field of bio-based functional fiber manufacturing, in particular to a preparation method of a modified spider silk protein enhanced lyocell fiber, which comprises the following steps: firstly, carrying out spider silk protein expression through a pichia pastoris strain GS115 / pPIC9K-spider silk gene; secondly, performing biomimetic mineralization treatment on spider silk protein, and performing ultrasonic-assisted mineralization to form a silicon dioxide coating layer; then preparing a spinning solution to realize uniform mixing of the mineralized spider silk protein and cellulose; coaxial spinning is carried out through micro-fluidic spinning equipment, and skin-core structure fibers are constructed; and finally inducing the formation of a beta-folding structure in an ethanol / water / calcium chloride coagulating bath. The process breaks through the technical bottlenecks of phase separation, heat sensitivity inactivation and uneven dispersion of spider silk protein and cellulose, reduces the production cost, and provides a high-performance fiber material for the high-end textile and biomedical fields.

The invention relates to the technical field of recombinant protein design and synthesis, and particularly discloses a recombinant spider silk protein and a synthesis method thereof. The recombinant spider silk protein provided by the invention is subjected to N-terminal and C-terminal structural domain replacement of different spider species and different spider silk types, intermediate functional domain repetitive sequence large fragment recombination and other optimization designs, and a series of recombinant spider silk proteins are obtained through a cell-free protein synthesis method and prokaryotic fermentation expression; compared with natural spider dragline silk protein or other existing recombinant spider silk protein, the spider silk protein has good composite mechanical performance and high temperature resistance, meanwhile, the molecular weight is small, the total expression quantity and soluble expression are remarkably improved, and a novel raw material and a design synthesis method of the recombinant spider silk protein are provided for development of high-performance biological materials.

The invention discloses a method for producing recombinant spider silk protein based on microalgae photosynthetic expression and non-chromatographic purification, the recombinant spider silk protein and application, and belongs to the technical field of recombinant spider silk protein production. The method comprises the following steps: designing a target recombinant spider silk protein structure, taking a spider dragline silk protein repetitive unit as a core, matching with an end-side elastin structural domain, inserting an amber termination codon TAG at a specified glycine site of the repetitive unit, and introducing non-natural amino acid containing a click chemical functional group by virtue of a gene codon extension technology; the method comprises the following steps: artificially synthesizing an optimized gene expression cassette, constructing a chlamydomonas reinhardtii expression system, synchronously constructing an unnatural amino acid insertion system, co-transforming the gene expression cassette and the unnatural amino acid insertion system into chlamydomonas reinhardtii, screening positive clones, carrying out photosynthetic culture induced expression, and carrying out pH-induced reversible phase separation in combination with magnetic nanoparticle click chemical non-chromatographic purification. Finally, the high-purity recombinant spider silk protein is prepared, and a technical basis is provided for industrialization of biological materials.

To provide an improved method for purification of recombinant spider silk protein, thereby enhancing solubility and increasing silk protein recovery yield.SOLUTION: Provided is a method for improving solubilization, extraction, and isolation of a recombinant spider silk protein using a salt and alcohol buffer. Provided is a method for solubilizing a recombinant spider silk protein from a host cell, comprising: providing a cell culture comprising a host cell, the host cell expressing a recombinant spider silk protein; recovering an insoluble portion derived from the cell culture, the insoluble portion comprising the recombinant spider silk protein; and adding the insoluble portion of the host cell to a solution comprising a salt and an alcohol, thereby solubilizing the recombinant spider silk protein in the solution.SELECTED DRAWING: Figure 1

The present application relates to the technical field of water treatment, and particularly relates to a water purifying agent based on a biomimetic adsorption principle and a preparation process thereof, wherein the water purifying agent is obtained by a composite framework of an inner layer, an intermediate layer and an outer layer; the inner layer is a polydopamine nanofiber network; the intermediate layer is a spider silk protein-paraffin composite layer; and the outer layer is a spider silk protein-polydopamine macroporous framework structure; through the multi-layer composite structure, the spider silk protein-polydopamine macroporous framework is the outer layer, the spider silk protein-paraffin composite layer is the intermediate layer, and the polydopamine nanofiber network is the inner layer, so that gradient capture of pollutants from micron to nanometer is realized, and through biomimetic tough framework design, dynamic pore size regulation mechanism, hierarchical adsorption synergistic effect and biomimetic adhesion enhancement technology, the limitation of traditional adsorbents is broken through.

The present application discloses silk improved by a spider silk protein gene and a method for using a spider silk protein gene to improve silk. The silk improved by the spider silk protein gene comprises a silk protein light chain. The silk protein light chain is linked to a spider silk protein which is formed by fusion expression with the silk fibroin light chain. An amino acid sequence of the spider silk protein comprises a repeat region sequence having at least one spider silk protein repeat unit sequence. The present application achieves the expression of Nephila pilipes pyriform silk in Bombyx mori silk fibroin, implements formation of an intact spider silk protein on the silk light chain, and expands sites for Bombyx mori silk gland gene editing. Light-chain gene edited Bombyx mori and a heavy-chain gene edited silkworm can be hybridized, thereby providing technical support for further selective breeding and optimization of fiber performance.

The invention discloses a recombinant expression and in-vitro splicing integrated preparation method and application of spider silk protein, and belongs to the technical field of biology. Coding genes of NT, Rep or CT regions of spider silk protein from at least two different spider species are fused, a fusion gene sequence is optimized by pichia pastoris codon and then introduced into an Intein tag, and the spider silk protein is obtained. Dividing the full-length protein into an N-terminal fragment and a C-terminal fragment; respectively cloning the N-terminal fragment and the Intein-N structural domain as well as the C-terminal fragment and the Intein-C structural domain into a pichia pastoris secreting type expression vector pPICZalpha; respectively electrically transforming the two obtained vectors into pichia pastoris SMD1168 for induced expression, and collecting supernatant; sequentially carrying out Ni-NTA affinity chromatography and ion exchange chromatography on the supernatant to obtain a purified N-terminal protein fragment and a purified C-terminal protein fragment; mixing the spider silk protein and the spider silk protein, forming full-length spider silk protein through Intein-mediated trans-splicing, and purifying through size exclusion chromatography. The method provided by the invention can realize high-efficiency and high-quality production of spider silk protein.

The invention belongs to the technical field of material modification, and particularly relates to a modified spider silk protein material and application thereof, specifically, the modified spider silk protein material comprises the following components in parts by weight: 25-35 parts of spider silk protein hydrolysate, 10-20 parts of chitosan hydrogel, 4-8 parts of pseudo-ginseng powder and 2-8 parts of glycerol. The gel wound dressing has a gel dosage form, is easy to coat, has good fluidity and good hydrophilicity, and has certain wound protecting and moisturizing effects, so that the recovery and reconstruction of the wound of a patient are facilitated.

The invention relates to a spider silk protein membrane material capable of generating electricity by friction and a preparation method and application thereof, a plurality of amino acid residues in flexible regions on two sides of a spider silk protein microcrystalline region are replaced with acidic amino acids or basic amino acids, and spider silk protein modifiers with different flexible region acidic / basic amino acid molar ratios are obtained; the spider silk protein modified body is independently formed or mixed with a conductive material to form a membrane, and the membrane material is formed. By utilizing the method disclosed by the invention, materials needing different piezoelectric properties can be developed, and a foundation is further laid for the industrial production of the recombinant spider silk protein and the wide application of the recombinant spider silk protein in the field of functional materials.

The invention relates to a recombinant spider silk protein fusion protein as well as a preparation method and application thereof. The fusion protein comprises a VNP6 tag and a recombinant spider silk protein, and the VNP6 tag is located at the N end of the fusion protein. A specific fusion protein structure is designed, and the intracellular space is regulated and controlled by using a VNP6 tag, so that the molecular congestion is effectively relieved, the protein expression yield is improved, and a brand new technical path is provided for improving the expression level of target protein, correctly folding and assembling supramolecular structures (such as artificial spider silk fibers, hydrogel and microneedles).

The application provides a cysteine-strengthened recombinant spider silk and a preparation method and application thereof. An amino acid sequence of the recombinant spider silk protein has the sequence shown in SEQ ID NO: 6. A polynucleotide encoding the amino acid sequence of the protein has the following structure: a 5' end of a cysteine-modified MaSp2 gene of the major ampullate gland of the spider Argiope bruennichi is connected with an N-domain, and a 3' end is connected with a C-domain; the gene is inserted into a pET-28a plasmid vector to obtain a recombinant plasmid, and the recombinant plasmid is introduced into E. coli to express the recombinant spider silk protein. The recombinant spider silk prepared by wet spinning using the recombinant spider silk protein has a dense beta-sheet crystal structure, excellent mechanical properties and wet strength, and opens up a prospect for industrial application of the recombinant spider silk.

The present invention relates to the technical field of recombinant protein design and synthesis, and specifically discloses a recombinant spider silk protein and its synthesis method. The recombinant spider silk protein provided by the present invention undergoes optimized design such as replacement of the N-terminal and C-terminal domains of different spider species and different spider silk types, and large fragment recombination of the middle functional domain repeat sequences. A series of recombinant spider silk proteins are obtained through cell-free protein synthesis methods and prokaryotic fermentation expression. Compared with natural spider dragline silk proteins or other existing recombinant spider silk proteins, they generally have good composite mechanical properties and high-temperature resistance characteristics. At the same time, they have a relatively small molecular weight, significantly improving the total expression level and soluble expression, providing new raw materials and design and synthesis methods for the development of high-performance biomaterials.

The invention discloses a spider silk protein transgene production method and application based on lentiviral transfection and sperm mediation in the field of biotechnology and genetic engineering. The spider silk protein transgene production method comprises the following steps: firstly, constructing a vector pLC-[3xP3-DsRed]-MaSp2-Amyloids for synthesizing and secreting spider silk protein genes by bombyx mori; the plasmid is introduced into a silkworm fertilized egg by using a lentivirus transfection and sperm-mediated gene transfer method, the red fluorescent protein gene and the black widow spider dragline silk protein 2 and amyloid polypeptide composite gene are introduced into a silkworm genome by using the characteristics of the lentivirus, and stable inheritance and expression are obtained; therefore, a transgenic silkworm capable of specifically synthesizing and secreting spider silk protein in bombyx mori silk gland cells is created, then the spider silk protein is synthesized and secreted by the silkworm, and the spider silk protein-containing composite silk with bombyx mori silk as the background is obtained. The method lays a foundation for mass production of black spider silk protein and also lays a foundation for improvement of mechanical properties of silk fibers.

To provide: improved methods for purifying recombinant spider silk polypeptides to increase the solubility and the silk protein recovery rate; and compositions comprising recombinant spider silk proteins.SOLUTION: A method of isolating a recombinant spider silk protein from a host cell is provided which comprises: a. providing an insoluble substance comprising a recombinant spider silk protein; b. adding the insoluble substance to an aqueous solution comprising a solvent; and c. applying a shear force to the aqueous solution comprising the insoluble substance thereby solubilizing the recombinant spider silk protein in the aqueous solution.SELECTED DRAWING: Figure 1A

The present application relates to the technical field of bio-based functional fiber manufacturing, in particular to a preparation method of modified spider silk protein reinforced lyocell fiber, comprising the following steps: firstly, spider silk protein is expressed by Pichia pastoris strain GS115 / pPIC9K-spider silk gene; secondly, the spider silk protein is subjected to biomimetic mineralization treatment, and a silicon dioxide coating layer is formed through ultrasonic-assisted mineralization; then, spinning solution is prepared to realize uniform mixing of the mineralized spider silk protein and cellulose; then, coaxial spinning is performed through a microfluidic spinning device to construct a skin-core structure fiber; finally, a beta-fold structure is induced to form in an ethanol / water / calcium chloride coagulation bath. The process breaks through the technical bottlenecks of phase separation, thermal inactivation and uneven dispersion of spider silk protein and cellulose, reduces production cost, and provides high-performance fiber materials for high-end textile and biomedical fields.

The invention relates to the technical field of biology, and provides a spider silk-spider venom protein fusion protein and a modified fiber, the spider silk-spider venom protein fusion protein comprises a spider silk protein or spider silk-like protein and a spider venom protein, and the spider silk-spider venom protein fusion protein is formed by fusion of the spider silk protein or spider silk-like protein and the spider venom protein. Researches find that spider silk proteins have high hydrophilicity and high water solubility and tend to mutually aggregate in a solution to generate liquid-liquid phase separation liquid drops, water-soluble expression of the proteins is greatly improved, the spider silk proteins are used as a functional protein carrier, spider venom proteins are fused on a spider silk protein framework to generate fusion proteins with high water solubility, and the spider silk proteins are used as functional protein carriers. Various soluble spider venom proteins can be produced through microbial fermentation, and the yield of the spider venom proteins is improved, for example, various soluble spider venom proteins can be produced through an escherichia coli expression system.