59 results about "Isopeptide bond" patented technology

The invention discloses a self-assembly enzyme for directional synthesis of uniform low molecular weight dextran, the self-assembly enzyme is named as YG-SpyTag-SpyCatcher-padex, the YG is a dextran sucrase gene (Genbank No.DQ345760), the padex is a dextran enzyme gene (Genbank No.KF999646.1), the SpyTag and the SpyCatcher are two specific oligopeptides, and aspartic acid in the SpyTag and lysine in the SpyCatcher spontaneously form an isopeptide bond. The invention belongs to the technical field of biology, and relates to an enzyme engineering technology. According to the invention, dextran sucrase derived from an escherichia coli genetically engineered bacterium BL21 (DE3)-YG-SpyTag and dextran enzyme derived from a pichia pastoris genetically engineered bacterium X33-SpyCatcher-padex are combined through a SpyTag-SpyCatcher protein covalent coupling system, and the obtained self-assembled enzyme can be used for catalyzing the substrate sucrose to be directly converted into dextran with the molecular weight of 3000 to 4000 Da.

The present invention relates to vaccine compositions, most particularly vaccine compositions wherein the antigenic component is large (e.g. greater than 50 kDa), or is multimeric (i.e. composed of subunits). Such antigenic components are of particular interest as they may represent antigenic components from pathogens against which vaccines are currently not available. The present invention relates to a composition comprising particles displaying an antigenic component, wherein the composition comprises an antigenic component comprising a first peptide tag and a portion comprising a second peptide tag, wherein the antigenic component and the portion are linked by an isopeptide bond between the first and second peptide tags, and wherein the antigenic component is greater than 50 kDa, or alternatively is multimeric.

The invention discloses a bacterial outer membrane vesicle protein drug delivery system derived from attenuated salmonella as well as a preparation method and application of the bacterial outer membrane vesicle protein drug delivery system. According to the bacterial outer membrane vesicle protein drug delivery system, plug-and-play of a protein drug can be realized on the surface of a bacterial outer membrane vesicle. The invention constructs a bacterial outer membrane vesicle protein drug delivery system. OmpA of attenuated salmonella typhimurium VNP20009 is knocked out, recombinant OmpA of SpyTag inserted into an extracellular ring structural domain is supplemented, fusion protein of SpyCatcher and a protein drug is displayed on the surface of the bacterial outer membrane vesicle by means of the property that an isopeptide bond is spontaneously formed between SpyTag and SpyCatcher, the preparation method is simple and easy to operate, various proteins are displayed on the surface of the bacterial outer membrane vesicle, and the bacterial outer membrane vesicle has a good application prospect. And protein drugs are efficiently protected and loaded. And by combining the immunostimulation characteristic of the bacterial outer membrane vesicles, efficient treatment of tumors is realized.

The invention discloses a recombinant I-type human collagen, hydrogel and application of the recombinant I-type human collagen and the hydrogel in preparation of a medical beauty filler. The recombinant I-type human collagen with high content of isopeptide bonds and nanocellulose are prepared into the composite hydrogel, and the composite hydrogel has the advantages of high stability, low swelling, high weight guarantee rate, strong adhesive force and hemostasis capability, and has the potential of being used as a skin tissue repair material. And the composite hydrogel can be used for repairing burnt skin, obviously reducing inflammatory response of the burnt skin and promoting wound healing. Besides, the composite hydrogel can also be used as a filler or an inhibitor to be filled in a mouse body, has relatively low inflammatory response, can promote collagen synthesis in the mouse body, can promote blood vessel and bone cell generation through long-term action, further promotes tissue neogenesis, and provides a technical basis for serving as a medical beauty filling material.

The invention relates to the technical field of biology, and particularly discloses a protein-loaded exosome as well as a preparation method and application thereof. The method for preparing the protein-loaded exosome comprises the following steps: (1) mixing a cholesterol-PEG-spy tag / probe tag with the exosome for reaction to obtain the spy tag / probe tag-loaded exosome; and (2) carrying out mixed reaction on the exosome loaded with the spy tag / probe tag and the fusion protein of the target loading protein and the spy catcher / probe, and forming an isopeptide bond through the spy tag / probe tag and the spy catcher / probe, so that the target loading protein is loaded on the exosome. The preparation method provided by the invention can avoid false positive, and the product is single and easy for mass production.

The invention relates to the technical field of biology, in particular to a mirror image coupled protein nanoparticle conjugate and application thereof. The conjugate comprises: ii) a functional protein labeled by a first peptide tag, and ii) a scaffold protein labeled by a second peptide tag, the first peptide tag and the second peptide tag being composed of D-amino acid residues; the first peptide tag and the second peptide tag form a peptide partner pair, and the first peptide tag and the second peptide tag are coupled and combined through an isopeptide bond. The conjugate provided by the invention is easier to escape from the recognition of protease, can obviously prolong the half-life period in a biological system, is not easy to cause unnecessary immunoreaction, and has good biological stability and safety.

The invention relates to molecular glue for forming fimbriae related protein based on SpaA isopeptide bonds of Vinci bacteria. The invention also relates to a nanoparticle vaccine composition formed by the molecular glue, and the immunogen is covalently displayed on the surface of the nanoparticle by the molecular glue.

The invention belongs to the technical field of enzyme engineering and gene engineering, and particularly relates to a recombinant immobilized enzyme, an expression gene thereof and application of the recombinant immobilized enzyme in production of macrocyclodextrin. According to the preparation method, SpyTag and SpyCatcher peptides are fused to the N end of isoamylase and the N end of 4-alpha-glucanotransferase respectively, the 4-alpha-glucanotransferase modified by the SpyTag and the isoamylase modified by the SpyCatcher are obtained, and after spontaneous isopeptide bonds are formed between the SpyTag and the SpyCatcher, the self-assembled macrocyclodextrin production enzyme cluster is obtained. The obtained macrocyclodextrin production enzyme cluster forms an enzyme aggregate with a three-dimensional structure under the traditional glutaraldehyde-mediated catalytic action, namely the recombinant immobilized enzyme. The recombinant immobilized enzyme disclosed by the invention can be repeatedly utilized, is simple and convenient to operate, is easy to separate a converted product, and can be applied to industrial production of the macrocyclodextrin.

The invention relates to a surface display system based on an isopeptide bond and a construction method and application of the surface display system, two conformational proteins including an anchor protein containing the isopeptide bond and a target protein are designed through a modularization method, and through covalent coupling formed between lysine and asparagine or aspartic acid residues in a molecular adhesive system based on the isopeptide bond, the surface display system based on the isopeptide bond is obtained. And displaying the target protein on the surface of escherichia coli. According to the surface display system based on the isopeptide bond, high-efficiency display of complex enzyme and macromolecular protein on the cell surface is realized, and the influence of direct fusion with anchoring protein on the activity of target protein is avoided. According to the constructed surface display whole-cell catalyst, the tedious enzyme purification process is omitted, the stability of the enzyme is improved, the production cost is lower, and the surface display whole-cell catalyst shows a good application prospect in biological catalysis.

The present invention relates to vaccine compositions, most notably vaccine compositions wherein the antigenic component is large, for example over 50 kDa, or multimeric, i.e. comprised of subunits. Such antigenic components are of particular interest, because they may represent antigenic components from pathogens that currently it is not possible to vaccinate against. The invention relates to a composition comprising a particle displaying an antigenic component, wherein said composition comprises an antigenic component comprising a first peptide tag, and a moiety comprising a second peptide tag, wherein the antigenic component and the moiety are linked via an isopeptide bond between said first and second peptide tags, and wherein the antigenic component is over 50 kDa, or alternatively is multimeric.

The present invention relates to a polypeptide forming one part of a two-part linker wherein the polypeptide (protein) spontaneously forms an isopeptide bond with a peptide tag, i.e. The other part of the two-part linker. The invention also relates to a method for producing a polypeptide of the invention and to the use of said polypeptide. According to the present invention, the reaction rate of the Sdy-Catcher variant obtained through mutation is significantly improved compared to the reaction rate of Sdy-Tag, such that the ideal direction is provided for the searching of polypeptides with characteristics of high binding rate and good binding effect in the field.

The invention discloses a preparation method and application of a high-strength, mildew-proof and low-cost cottonseed meal protein adhesive. The preparation method comprises the following steps: compounding L-lysine and 4-carboxyphenylboronic acid through a coupling reaction to prepare lysine borate, adding the prepared lysine borate into the cottonseed meal protein adhesive, adding a proper amount of transglutaminase, and uniformly stirring to obtain the novel high-strength, mildew-proof and low-cost cottonseed meal protein adhesive based on the skin bionic strategy. The innovation points of the invention are as follows: by simulating a peptide bond between protein and polysaccharide in skin and forming a compact cross-linked layer through dehydration condensation reaction, using transglutaminase to construct a grain-lysine isopeptide bond, and introducing boric acid to form a covalent boron-oxygen-six-ring structure, a dual cross-linked network is constructed. The cottonseed meal protein adhesive has the advantages that the bonding strength, mildew resistance and other properties of the cottonseed meal protein adhesive can be remarkably improved only by using trace transglutaminase and boron oxygen six-ring as cross-linking agents, and the preparation cost of the adhesive is reduced.

The invention relates to a molecular peptide mutant, the amino acid sequence of which is as shown in SEQ ID NO: 1. In the invention, SpyCatcher is designed and modified to obtain a molecular peptide SpyCatcher-21 with stimulus response to pH on the basis of not affecting the formation of isopeptide bonds, and Pro is introduced into a key loop of the SpyCatcher-21 through analysis of the crystal structure to reduce the flexibility of the loop and obtain a mutant SpyCatcher-21_A82P, which can raise ligation efficiency with SpyTag. The SpyCatcher-21_A82P can be used to achieve double-enzyme catalysis according to objective needs by changing the pH of the environment to obtain different degrees of coupling, or obtain a three-enzyme coupled catalytic system through electrostatic interaction with a positively charged enzyme.

The invention discloses a SpaD-based polypeptide-protein reaction pair, and belongs to the technical field of protein engineering. The D2-D3 structural domain of the corynebacterium diphtheriae pilus protein SpaD is split into reaction pairs in two modes, the two parts can be efficiently recombined and react to generate isopeptide bonds, and covalent coupling is achieved. Wherein when the D2 structural domain is split into reaction pairs, a D214-T455 residue of an original sequence is a capturer protein (D2C), and an E182-K213 residue of the original sequence is a tag polypeptide (D2T); when the D3 structural domain is split into reaction pairs, the E182-D434 residue of the original sequence is a capturer protein (D3C), and the G438-T455 residue of the original sequence is a tag polypeptide (D3T). The reactivity of a reaction pair obtained by splitting D3 is equivalent to that of a common spy reaction pair, is not influenced by the position of a tag sequence, is orthogonal to the spy reaction pair and a probe reaction pair, is a novel protein coupling tool, and has a relatively high practical value in protein engineering.

The present disclosure provides a heterodimeric antibody, or antigen-binding fragment thereof, comprising two heavy chains with different amino acid sequences and a binding polypeptide. The heterodimer antibody, or the antigen-binding fragment thereof, include a first heavy chain comprising none or one or more modifications and the second heavy chain comprises one or more modifications and is fused to a binding polypeptide, which can form a isopeptide bond with a binding peptide. The present disclosure further provides systems and methods for purifying such heterodimeric antibodies, and antigen-binding fragments thereof.

The present invention concerns Gram-negative bacterial cells or Outer Membrane Vesicles (OMVs) which display on their outer surface an autotransporter (AT) fusion protein covalently coupled via an isopeptide bond to a heterologous molecule. These bacterial cells and OMVs are suitable for use in vaccines or targeted drug delivery of antigens or therapeutic agents to specific cells or tissues.

This invention discloses a method for rapidly constructing a complex of cholera toxin B subunit (CTB) and antigen. The vaccine complex includes a CTB-SpyTag fusion protein and at least one SpyCatcher-antigen fusion protein. It also includes a preparation method for rapidly preparing a CTB5-antigen complex by mixing CTB-SpyTag with the SpyCatcher-antigen. This invention directs the antigen to the side of the CTB pentamer that does not participate in GM1 binding, avoiding steric hindrance interference. It utilizes the SpyTag / SpyCatcher covalent isopeptide bond to obtain highly stable antigen display, reducing antigen shedding during subsequent preparation and storage. It also possesses mucosal targeting and multivalent presentation functions, providing a universal platform technology for rapidly constructing and validating broad-spectrum, combined, or personalized mucosal vaccines.

This invention provides a recombinant Staphylococcus aureus enterotoxin B nanoparticle protein vaccine, mSEB-mi3, its preparation method, and its applications. This invention utilizes a SpyTag / SpyCather protein linker system to connect the recombinant protein to the nanoparticle protein, allowing mSEB to be displayed on the surface of self-contained nanoparticles, thus obtaining the recombinant Staphylococcus aureus enterotoxin B nanoparticle protein mSEB-mi3. Both mSEB-SpyTag and SpyCather-mi3 proteins in this invention are induced to be expressed in soluble form in Escherichia coli. The mSEB-SpyTag and SpyCather-mi3 of this invention can be covalently bound via isopeptide bonds to form the mSEB-mi3 nanoparticle protein vaccine. The binding conditions are simple, and when combined with the adjuvant MF59, it can induce rapid antibody production in mice, generating a high-level immune response and exerting a protective effect. Furthermore, its antibody response rate and protective efficiency are superior to those of the monomeric protein mSEB.

The invention is suitable for the technical field of gene engineering, and provides 3Ag + mi3 protein nanoparticles for preventing and treating BVD and IBR as well as a preparation method and application of the 3Ag + mi3 protein nanoparticles, the 3Ag + mi3 protein nanoparticles comprise E2-1b-st protein, E2-1d-st protein, gD-st protein and mi3-sc protein; the E2-1b-st protein, the E2-1d-st protein and the gD-st protein are coupled to the surface of the same mi3-sc protein through an isopeptide bond to form 3Ag + mi3 protein nanoparticles; the amino acid sequences of the E2-1b-st protein, the E2-1d-st protein, the gD-st protein and the mi3-sc protein are respectively shown as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 in a sequence table. After animals are immunized by the 3Ag + mi3 protein nanoparticles, an effective immune protection effect can be generated, and the 3Ag + mi3 protein nanoparticles have a good prevention and control application prospect on bovine viral diarrhea and bovine rhinotracheitis.

The present invention relates to a polypeptide that forms one part of a two-part linker in which the polypeptide spontaneously forms an isopeptide bond with a peptide tag, the second part of the two-part linker. Nucleic acid molecules encoding the polypeptide, vectors comprising said nucleic acid molecules, and host cells comprising said vectors and nucleic acid molecules are also provided. A kit comprising said two-part linker (i.e. peptide tag and polypeptide binding partner), and / or nucleic acid molecules / vectors is also provided. A method of producing the polypeptide (binding partner) and the uses of the polypeptide of the invention are also provided.

The present disclosure relates to the field of cell-free protein or polypeptide production for the purpose of producing a polypeptide of interest (POI) which comprises a moiety, which is a target polypeptide whereof production is desired. The target polypeptide is linked to a coupling peptide which binds to a complementary peptide which is fused to an autotransporter fusion protein which is displayed on the outer surface of a vesicle such that the synthesized POI is coupled to said vesicle via isopeptide bond. Furthermore, the disclosure relates to compositions comprising such a target polypeptide moiety, which are amenable to use in therapeutic applications and / or biomedical applications.

This invention provides a recombinant Staphylococcus aureus nanoparticle protein HI-mi3, its preparation method, and its applications. This protein is composed of HI (containing the key SA virulence factor α-hemolysin Hla). H35L The fusion protein HI and mi3 are composed of the N2 functional domain of the iron ion surface determinant B and self-assembled nanoparticle protein mi3. This invention uses the SpyTag / SpyCatCher protein coupling system to link the fusion protein antigen HI and mi3, obtaining the Staphylococcus aureus self-assembled nanoparticle protein HI-mi3. Both the recombinant protein SpyCatcher-mi3 and the recombinant protein SpyTag-HI of this invention are induced to be expressed in soluble form in Escherichia coli. The recombinant protein SpyCatcher-mi3 of this invention can covalently bind to the recombinant protein SpyTag-HI via isopeptide bonds to form the HI-mi3 nanoparticle vaccine molecule, with simple binding conditions. The HI-mi3 nanoparticle vaccine of this invention can induce a high level of immune response in animals and exert an immunoprotective effect.

The present invention discloses a self-assembling enzyme for the directed synthesis of uniform low-molecular-weight dextran, named YG-SpyTag-SpyCatcher-padex. YG represents the dextransucrase gene (GenBank No. DQ345760), padex represents the dextranase gene (GenBank No. KF999646.1), SpyTag and SpyCatcher represent two specific short peptides, and the aspartic acid in SpyTag spontaneously forms an isopeptide bond with the lysine in SpyCatcher. The present invention belongs to the field of biotechnology and relates to enzyme engineering technology. The present invention combines dextran sucrase derived from the genetically engineered Escherichia coli bacterium BL21(DE3)-YG-SpyTag with dextranase derived from the genetically engineered Pichia pastoris bacterium X33-SpyCatcher-padex through the SpyTag-SpyCatcher protein covalent coupling system. The obtained self-assembling enzyme can catalyze the direct conversion of the substrate sucrose into dextran with a molecular weight of 3000-4000Da.

The application discloses an immunological composition containing a fatty acid modified SpyCatcher protein and a preparation method thereof, and belongs to the field of biological medicines; wherein the SpyCatcher protein (LipoSC) can self-polymerize into nanoparticles, and covalent connection with an antigen containing a SpyTag polypeptide through an isopeptide bond can improve the immunogenicity of the loaded antigen; taking the receptor binding domain (RBD) of the coronavirus spike protein (S protein) as an example, the immunological composition containing the fatty acid modified SpyCatcher protein and the coronavirus RBD antigen disclosed by the application can induce a strong RBD specific immune response, and in particular, as a mucosal subunit vaccine, can induce antigen specific mucosal immunity and humoral immunity. The application further provides a method for realizing the fatty acid modification of the SpyCatcher protein in Escherichia coli, and a method for preparing various immunological compositions by using the fatty acid modified protein.

The invention belongs to the technical field of biology, and relates to a semi-synthetic nanopore, a preparation method thereof and a protein sequencing method. The semi-synthetic nanopore comprises a polypeptide binding protein and a construction body of a nanopore channel, and the polypeptide binding protein can unfold protein into linear polypeptide binding protein; in addition, the polypeptide binding protein is combined with the nanopore channel through an amido bond; preferably, the binding by amido bonds comprises the binding by transpeptidase properties or isopeptide bonds; preferably, the polypeptide binding protein is a folding enzyme; preferably, the nano pore channel is a nano pore channel protein. The current characteristic spectrum with the time sequence when the protein full-length sequence penetrates through the nanopore is obtained, and the problem that the reading length of existing nanopore protein sequencing is short is solved.

The invention provides an engineered probiotic-polymer conjugate and application thereof, SpyCatcher protein is displayed on the surface of probiotics, a polymer comprises a SpyTag residue and a dopamine structure part, and the probiotics and the polymer are coupled through an isopeptide bond formed between the SpyCatcher and the SpyTag. The colonization ability of the conjugate in the gastrointestinal tract is remarkably improved, the tolerance of the probiotics in the gastrointestinal tract is effectively improved, the construction method of the conjugate does not affect the inherent probiotic activity of the probiotics, and the conjugate has important significance in treatment of gastrointestinal diseases and is expected to promote the development of engineered intestinal microorganisms.

The invention is applicable to the technical field of genetic engineering, and provides an E2-1b + mi3 protein nanoparticle of bovine viral diarrhea virus as well as a preparation method and application of the E2-1b + mi3 protein nanoparticle, the E2-1b + mi3 protein nanoparticle is formed by coupling an E2-1b-st fusion protein and a mi3-sc fusion protein, the 1b type E2 antigen of the bovine viral diarrhea virus is displayed on the surface of the mi3 protein nanoparticle through an isopeptide bond; the amino acid sequence of the mi3 protein nanoparticle is shown as SEQ ID NO: 6 in a sequence table; the amino acid sequence of the E2-1b-st fusion protein is as shown in SEQ ID NO: 2 in a sequence table; the amino acid sequence of the mi3-sc fusion protein is shown as SEQ ID NO: 4 in a sequence table. After animals are immunized by the E2-1b + mi3 protein nano-particles, an effective immune protection effect can be generated, and the E2-1b + mi3 protein nano-particles have a good prevention and control application prospect on bovine viral diarrhea caused by BVDV 1b type viruses.

The invention provides a nanoparticle protein rePO-TTFC (at) LS as well as a preparation method and application thereof. The protein is composed of rePO (at) LS and a tetanus toxin C fragment TTFC. The recombinant protein SpyCatcher-rePO at LS disclosed by the invention is subjected to induced expression in escherichia coli in a soluble form. According to the present invention, the recombinant protein SpyCatcher-rePO (at) LS can be subjected to covalent binding with the recombinant protein reTTFC-SpyTag through the isopeptide bond so as to form the rePO-TTFC (at) LS duplex nanoparticle vaccine molecule, and the binding condition is simple; the nanoparticle protein can induce an animal body to generate high-level immune response and play an immune protection role, the response speed and protection efficiency of an antibody induced by the nanoparticle protein are obviously superior to those of a monomer rePO and a monomer reTTFC fusion protein, and the nanoparticle protein can be used for preventing and treating pseudomonas aeruginosa and tetanus at the same time.

The invention belongs to the field of protein hydrogel materials, and particularly relates to elastin-like and a preparation method and application of elastin-like hydrogel. According to the present invention, the two elastin-like proteins A and B having different amino acid sequences are utilized to form the cross-linked network through the isopeptide bond, such that the mechanical strength of the polymer hydrogel composed of the four-arm polyethylene glycol amine, 4-arm PEG-NH2 and the o-phthalaldehyde modified four-arm polyethylene glycol, 4-arm PEG-OPA can be effectively improved, the adhesion can be improved, and the cross-linked network can be used for the preparation of the polymer hydrogel, such that the cross-linked network can be formed through the cross-linked network, such that the cross-linked network can be used for the preparation of the polymer hydrogel, and the use amount of the polymer hydrogel can be reduced. The obtained elastin-like hydrogel has the advantages of strong wet tissue adhesion capacity, good biocompatibility, injectable use and the like, and is beneficial to application in the biomedical fields of hemostasis, tissue repair and the like.

To provide viral vector systems that maintain the integrity of a modified viral structure while remaining adaptable for targeted transfer of nucleic acids of interest to a variety of target cells.SOLUTION: Described herein is a viral retargeting strategy that solves the problems inherent in previous retargeting strategies by utilizing a first member and a second cognate member of a specific binding pair, where the first member and the second cognate member specifically interact to form a chemical, preferably covalent, bond. The first member, when displayed on a capsid protein, acts as a scaffold for any targeting ligand fused to the second cognate member, but upon binding of the first member and the second cognate member, an isopeptide bond forms and recombinant viral particles act as a one-component targeting vector.SELECTED DRAWING: None