79 results about "Sortase" patented technology

Methods for the in vitro production of enucleated red blood cells and the enucleated red blood cells thus prepared are provided. Such enucleated red blood cells may express a sortaggable surface protein, which allows for surface modification in the presence of a sortase. Also described herein are surface modified enucleated red blood cells, e.g., conjugated with an agent of interest such as a peptide, a detectable label, or a chemotherapeutic agent, and uses thereof in delivering the agent to a subject.

The present invention is a substantially purified sortase-transamidase enzyme from Gram-positive bacteria, such as Staphylococcus aureus. A specific sortase-transamidase enzyme disclosed has a molecular weight of about 29,076 daltons and catalyzes a reaction that covalently cross-links the carboxyl terminus of a protein having a sorting signal to the peptidoglycan of a Gram-positive bacterium, where the sorting signal has a a motif of NPQ / KTN / G therein. Variants of the enzyme, methods for cloning the gene encoding the enzyme and expressing the cloned gene, and methods of use of the enzyme, including for screening for antibiotics and for display of proteins or peptides on the surfaces of Gram-positive bacteria, are also disclosed.

Strategies, systems, methods, reagents, and kits for the directed evolution of bond-forming enzymes are provided herein. Evolution products, for example, evolved sortases exhibiting enhanced reaction kinetics and / or altered substrate preferences are also provided herein, as are methods for using such evolved bond-forming enzymes. Kits comprising materials, reagents, and cells for carrying out the directed evolution methods described herein are also provided.

The present invention provides methods and compositions involving sortase-transamidases, including sortase B, and polypeptides that include a signal sorting sequence of NPQ / KTN / G. Methods of screening for inhibitors of Gram-positive bacteria as well as therapeutic, preventative, and research methods focusing on Gram-positive bacteria are also provided.

The present invention is a substantially purified sortase-transamidase enzyme from Gram-positive bacteria, such as Staphylococcus aureus. The enzyme having a molecular weight of about 23,539 or about 29,076 daltons and catalyzing a reaction that covalently cross-links the carboxyl terminus of a protein having a sorting signal to the peptidoglycan of a Gram-positive bacterium, the sorting signal having: (1) a motif of LPX3X4G therein; (2) a substantially hydrophobic domain of at least 31 amino acids carboxyl to the motif; and (3) a charged tail region with at least two positively charged residues carboxyl to the substantially hydrophobic domain, at least one of the two positively charged residues being arginine, the two positively charged residues being located at residues 31-33 from the motif, wherein X3 is any of the twenty naturally-occurring L-amino acids and X4 is selected from the group consisting of alanine, serine, and threonine, and wherein sorting occurs by cleavage between the fourth and fifth residues of the LPX3X4G motif. Variants of the enzyme, methods for cloning the gene encoding the enzyme and expressing the cloned gene, and methods of use of the enzyme, including for screening for antibiotics and for display of proteins or peptides on the surfaces of Gram-positive bacteria, are also disclosed.

Strategies, systems, methods, reagents, and kits for the directed evolution of bond-forming enzymes are provided herein. Evolution products, for example, evolved sortases exhibiting enhanced reaction kinetics and / or altered substrate preferences are also provided herein, as are methods for using such evolved bond-forming enzymes. Kits comprising materials, reagents, and cells for carrying out the directed evolution methods described herein are also provided.

In some aspects, polypeptides comprising single domain antibodies and methods of identifying single domain antibodies are provided. In some embodiments polypeptides comprising a single domain antibody and a sortase recognition sequence, are provided. In some aspects, products and methods of use in modulating the immune system, e.g., modulating an immune response, are provided.

The present invention is a substantially purified sortase-transamidase enzyme from Gram-positive bacteria, such as Staphylococcus aureus. The enzyme having a molecular weight of about 23,539 or about 29,076 daltons and catalyzing a reaction that covalently cross-links the carboxyl terminus of a protein having a sorting signal to the peptidoglycan of a Gram-positive bacterium, the sorting signal having: (1) a motif of LPX3X4G therein; (2) a substantially hydrophobic domain of at least 31 amino acids carboxyl to the motif; and (3) a charged tail region with at least two positively charged residues carboxyl to the substantially hydrophobic domain, at least one of the two positively charged residues being arginine, the two positively charged residues being located at residues 31-33 from the motif, wherein X3 is any of the twenty naturally-occurring L-amino acids and X4 is selected from the group consisting of alanine, serine, and threonine, and wherein sorting occurs by cleavage between the fourth and fifth residues of the LPX3X4G motif. Variants of the enzyme, methods for cloning the gene encoding the enzyme and expressing the cloned gene, and methods of use of the enzyme, including for screening for antibiotics and for display of proteins or peptides on the surfaces of Gram-positive bacteria, are also disclosed.

The present invention relates to methods and compositions for use in modulating, including inhibiting the growth and / or reducing the virulence of gram-positive bacteria. The present invention provides methods and compositions for disrupting the cell wall and / or cell membrane in gram-positive bacteria such that cell wall or cell membrane target(s) are rendered exposed or accessible and sensitive to a modulation thereof. Methods for modulation of one or more gram-positive bacterial cell wall or cell membrane targets in a gram-positive bacteria are provided comprising disrupting the cell wall such that the cell wall or cell membrane target, which is particularly a sortase, is rendered exposed or accessible and sensitive to a modifying, modulating or binding agent, which is particularly an antibody or fragment thereof, wherein the cell wall or cell membrane target is inaccessible or relatively insensitive to the modifying, modulating or binding agent in the absence of cell wall disruption.

Methods for the in vitro production of enucleated red blood cells and the enucleated red blood cells thus prepared are provided. Such enucleated red blood cells may express a sortaggable surface protein, which allows for surface modification in the presence of a sortase. Also described herein are surface modified enucleated red blood cells, e.g., conjugated with an agent of interest such as a peptide, a detectable label, or a chemotherapeutic agent, and uses thereof in delivering the agent to a subject.

The present invention is a substantially purified sortase-transamidase enzyme from Gram-positive bacteria, such as Staphylococcus aureus. A specific sortase-transamidase enzyme disclosed has a molecular weight of about 29,076 daltons and catalyzes a reaction that covalently cross-links the carboxyl terminus of a protein having a sorting signal to the peptidoglycan of a Gram-positive bacterium, where the sorting signal has a a motif of NPQ / KTN / G therein. Variants of the enzyme, methods for cloning the gene encoding the enzyme and expressing the cloned gene, and methods of use of the enzyme, including for screening for antibiotics and for display of proteins or peptides on the surfaces of Gram-positive bacteria, are also disclosed.

The invention relates to a method for catalyzing L-glutamine and ethylamine to generate L-theanine by using recombinant Bacillus subtilis secreted gamma-glutamyltranspeptidase and belongs to the field of genetic engineering and enzyme engineering. The method provided by the invention is used for amplifying genes of gamma-glutamyltranspeptidase (GGT) in bacillus subtilis and cloning overexpression in bacillus subtilis 168. According to the method, a bacillus subtilis engineering strain capable of secreting gamma-glutamyltranspeptidase is established for the first time and the enzyme activity of the strain and the fermenting property of enzyme are researched: the supernatant enzyme activity of recombinant bacteria reaches 49.8 U / mg which is 20 times of that of an original strain. By using 80mML-glutamine as a donor and 640mMZ ethylamine as a receptor, the conversion ratio of L-theanine can reach over 86%. The method provided by the invention uses the safe strain, has the advantages of being high in conversion ratio, simple in production method and the like, and facilitates production of L-theanine produced by an industrial scaled enzymic method.

The invention relates to bacterial cell surface display of post-translationally modified heterologous proteins. Provided is an isolated nucleic acid construct encoding a proteinaceous substance comprising, from the N-terminus to the C-terminus, at least (a) an N-terminal a lantibiotic leader sequence; (b) an amino acid sequence of interest to be post-translationally modified to a dehydroresidue- or thioether-bridge containing polypeptide; (c) a hydrophilic cell-wall spanning domain; (d) a sortase recognition motif; (e) a hydrophobic membrane spanning domain and (f) a C-terminal charged membrane anchoring domain. Also provided is a Gram-positive host cell expressing the construct, as well as a library of host cells.

The present invention relates to methods and compositions for use in modulating, including inhibiting the growth and / or reducing the virulence of gram-positive bacteria. The present invention provides methods and compositions for disrupting the cell wall and / or cell membrane in gram-positive bacteria such that cell wall or cell membrane target(s) are rendered exposed or accessible and sensitive to a modulation thereof. Methods for modulation of one or more gram-positive bacterial cell wall or cell membrane targets in a gram-positive bacteria are provided comprising disrupting the cell wall such that the cell wall or cell membrane target, which is particularly a sortase, is rendered exposed or accessible and sensitive to a modifying, modulating or binding agent, which is particularly an antibody or fragment thereof, wherein the cell wall or cell membrane target is inaccessible or relatively insensitive to the modifying, modulating or binding agent in the absence of cell wall disruption.

The invention discloses an electrochemical detection method of Staphylococcus aureus. The method comprises the following steps: preparing an ordered mesoporous carbon nano material CMK-3 by a template process, and carrying out surface carboxyl functionalization modification to obtain a carboxyl-functionalized mesoporous carbon nano material O-CMK-3; immobilizing sortase A (Srt A) onto the carboxyl-functionalized mesoporous carbon nano material O-CMK-3 by a chemical covalent binding technique to obtain an immobilized sortase nano composite material O-CMK-3-M; and preparing a carbon paste electrode from the immobilized sortase nano composite material, graphite, paraffin and other materials, immersing the carbon paste electrode into Staphylococcus aureus solutions with different concentrations, and detecting Staphylococcus aureus by using a tri-electrode detection system. Compared with the existing detection technique, the method has the advantages of short detection time and the like, is convenient to operate, is recyclable, and thus, has favorable application prospects.

The invention belongs to the technical field of biological engineering, and discloses a protein designated PEG modification method and an obtained PEG modified protein. The protein designated PEG modification method of the invention is as below: acquiring a protein, fusing a sorting motif recognized by a sortase at the C terminal of the protein to obtain a recombinant protein; mixing a PEG modifier, the recombinant protein and sortase, conducting peptide bond cutting and connection reaction to obtain a PEG modified protein, wherein the PEG modifier has a structure shown in a formula (I). The invention utilizes the sortase to realize designated PEG modification at the C terminal of the protein; the whole operation is simple and low in cost, avoids multisite modification in protein PEG modification, is more conducive to application of PEG modification in protein modification; m represents the degree of polymerization, and satisfies the relation of 1<=m<= 5; and n represents the degree of polymerization, and satisfies the relation of 110<= n<=1100.

The invention discloses a method for measuring sortase based on pichia pastoris surface display, comprising the following steps: A. infusing target gene QALPETGEE and linker-eGFP gene into QALPETGEE-linker-eGFP; B. constructing an expression carrier for constructing QALPETGEE-linker--eGFP; C. converting the expression carrier pKFS-QALPETGEE-linker-eGFP into pichia pastoris; D. expressing yeast surface protein: inoculating and culturing positive transformant by YPD and BGMY liquid culture medium, and inducing expression in BMMY liquid culture medium; E. measuring the yeast surface protein; F. expressing sortase; and G. testing activatedly the sortase. The invention uses the yeast surface display technology, fluorospectrophotometer and flow cytometry are adopted for measuring, and the used materials are lower in price, thus greatly reducing the measuring cost of sortase and facilitating operation.

The invention relates to a sortase-mediated protein purification and ligation. Specifically, the invention relates to a technique that links protein expression / purification with conjugation to therapeutic agents, imaging agents, or linkers.

Provided are improved methods for identifying the substrate recognition specificity or activity of a protease, convertase (sortase), or kinase. In some embodiments, methods are provided for identifying the endogenous protease or convertase cleaving patterns (e.g., “cleaveOme”) inside the secretory pathway of a living cell. Select embodiments involve aspects of yeast endoplasmic reticulum sequestration screening and next generation sequencing. Methods of producing polypeptides in Kex2 knockout yeast are also provided.

Methods and reagents for the installation of click chemistry handles on target proteins are provided, as well as modified proteins comprising click chemistry handles. Further, chimeric proteins, for example, bi-specific antibodies, that comprise two proteins conjugated via click chemistry, as well as methods for their generation and use are disclosed herein.

Methods for the in vitro production of enucleated red blood cells and the enucleated red blood cells thus prepared are provided. Such enucleated red blood cells may express a sortaggable surface protein, which allows for surface modification in the presence of a sortase. Also described herein are surface modified enucleated red blood cells, e.g., conjugated with an agent of interest such as a peptide, a detectable label, or a chemotherapeutic agent, and uses thereof in delivering the agent to a subject.

Provided herein are methods, nucleic acids, polypeptides, compositions, and kits relating to the conjugation of a heterologous polypeptide to a molecule of interest during production of the polypeptide in cell culture. In various embodiments, the heterologous polypeptide is linked to a sortase ligation sequence and the molecule of interest is linked to a complementary sortase ligation sequence, such that expression of the heterologous protein in the presence of the molecule of interest and cells expressing a surface-associated sortase with the sortase catalytic domain exposed to the extracellular medium results in ligation of the heterologous polypeptide to the molecule of interest to form a conjugated polypeptide.

In various embodiments a system is provided that displays heterologous proteins on the surface of a Gram-positive microorganism. In certain embodiments the system displays proteins using a sortase transpeptidase to covalently anchor proteins to the cell wall of the microbe. Novel bacterial strains are provided to exploit this system to display cellulase enzymes and multi-enzyme complexes on the surface of Gram-positive microorganisms (e.g., Bacillus subtilis) through their non-covalent interaction with a scaffoldin protein that is covalently anchored to the cell wall by the sortase transpeptidase. The surface displayed protein complexes contain enzymes capable of degrading cellulose into its component sugars at accelerated rates as compared to solutions of purified enzymes.

The invention relates to proximity-based sortase-mediated protein purification and ligation. Specifically, the invention relates to techniques that links protein expression / purification with conjugation to therapeutic agents, imaging agents, or linkers.

Methods of forming, dissolving, and functionalizing an extracellular matrix gel on demand based on cross-linking, modification, and dissolution of hydrogels using transpeptidase (e.g. sortase) are disclosed. Also provided are hydrogels comprising one or more macromers crosslinked to a mixture of peptides, wherein all or a portion of the peptides in the mixture comprise a recognition motif cleavable by a transpeptidase (e.g., sortase).

The invention relates to a fusion protein and use thereof. The fusion protein comprises, from N to C terminals, a first moiety, an Fc segment, a linker moiety comprising a moiety selected from the group consisting of a linker and a protein or polypeptide selected from the group consisting of IL2 or scFv, and a substrate moiety of transpeptidase A; the linker comprises a sequence selected from thegroup consisting of (1) (GGGGS) n, wherein when the linker moiety comprises the protein or polypeptide and linker, n>=1; when the linker moiety only comprises the joint, n>=3; and (2) (EAAK) n, n >= 1; the substrate moiety comprises a sequence as shown in LPXTG. The fusion protein can be directly connected to cells to enable the cells to have targeting property, is simpler than an existing methodfor preparing targeting cells through cell transfection, and can also reduce the risk possibly generated by effector cell genome operation at the same time.

Production of protein conjugate vaccines by use of transpeptidase enzymes, such as sortase enzymes. For example, homogenous immunoconjugates (e.g., a population of molecules having the same structure) formed by conjugating an antigenic polypeptide and a bacterial capsule component are provided. In certain aspects, methods for generating an immune response to B. anthracis by use of protective antigen-PDGA immunoconjugates are provided.