1044 results about "Disulfide bonding" patented technology

Disclosed are methods of producing eukaryotic disulfide bond-containing polypeptides in bacterial hosts, and compositions resulting therefrom. Co-expression of a eukaryotic foldase and a disulfide bond-containing polypeptide in a bacterial host cell is demonstrated. In particular embodiments, the methods have been used to produce mammalian pancreatic trypsin inhibitor and tissue plasminogen activator (tPA) in soluble, biologically-active forms, which are isolatable from the bacterial periplasm. Also disclosed are expression systems, recombinant vectors, and transformed host cells.

Disclosed are methods and compositions for producing heterologous disulfide bond containing polypeptides in bacterial cells. In preferred embodiments the methods involve co-expression of a prokaryotic disulfide isomerase, such as DsbC or DsbG and a gene encoding a recombinant eukaryotic polypeptide. Exemplary polypeptides disclosed include tissue plasminogen activator.

The invention relates to a method of preparing heteromultimeric polypeptides such as bispecific antibodies, bispecific immunoadhesins and antibody-immunoadhesin chimeras. The invention also relates to the heteromultimers prepared using the method. Generally, the method provides a multispecific antibody having a common light chain associated with each heteromeric polypeptide having an antibody binding domain. Additionally the method further involves introducing into the multispecific antibody a specific and complementary interaction at the interface of a first polypeptide and the interface of a second polypeptide, so as to promote heteromultimer formation and hinder homomultimer formation; and / or a free thiol-containing residue at the interface of a first polypeptide and a corresponding free thiol-containing residue in the interface of a second polypeptide, such that a non-naturally occurring disulfide bond is formed between the first and second polypeptide. The method allows for the enhanced formation of the desired heteromultimer relative to undesired heteromultimers and homomultimers.

The invention relates to a method of preparing heteromultimeric polypeptides such as bispecific antibodies, bispecific immunoadhesins and antibody-immunoadhesin chimeras. The invention also relates to the heteromultimers prepared using the method. Generally, the method provides a multispecific antibody having a common light chain associated with each heteromeric polypeptide having an antibody binding domain. Additionally the method further involves introducing into the multispecific antibody a specific and complementary interaction at the interface of a first polypeptide and the interface of a second polypeptide, so as to promote heteromultimer formation and hinder homomultimer formation; and / or a free thiol-containing residue at the interface of a first polypeptide and a corresponding free thiol-containing residue in the interface of a second polypeptide, such that a non-naturally occurring disulfide bond is formed between the first and second polypeptide. The method allows for the enhanced formation of the desired heteromultimer relative to undesired heteromultimers and homomultimers.

A single chain T cell receptor (scTCR) comprising an a segment constituted by a TCR α chain variable region sequence fused to the N terminus of a TCR α chain constant region extracellular sequence, a β segment constituted by a TCR β chain variable region fused to the N terminus of a TCR β chain constant region extracellular sequence, and a linker sequence linking the C terminus of the α segment to the N terminus of the β segment, or vice versa, the constant region extracellular sequences of the α and β segments being linked by a disulfide bond, the length of the linker sequence and the position of the disulfide bond being such that the variable region sequences of the α and β segments are mutually orientated substantially as in native αβ T cell receptors. Complexes of two or more such scTCRs, and use of the scTCRs in therapy and in various screening applications are also disclosed.

Dithioamine reducing agents useful for the reduction of disulfide bonds. The reducing agents of this invention are useful, for example, to reduce disulfide bonds, particularly in proteins, or to prevent the formation of disulfide bonds, particularly in proteins and other biological molecules. Reducing agents of this invention are useful and suitable for application in a variety of biological applications, particularly as research and synthetic reagents. The invention provides S-acylated dithioamines which can be selectively activated reducing agents by removal of the S-acyl groups enzymatically or chemically. The invention further provides dithiane precursors of thioamino reducing agents. The invention provides dithioamine reducing agents, S-acylated dithioamines and dithianes which are immobilized on surfaces, including among others, glass, quartz, microparticles, nanoparticles and resins.

The present invention provides methods for producing humanized antibodies and increasing the yield of antibodies and / or antigen binding fragments when produced in cell culture. In one aspect of the invention, at least one framework region amino acid residue of the variable domain is substituted by a corresponding amino acid from a variable domain consensus sequence subgroup that has the most sequence identity with the HVR1 and / or HVR2 amino acid sequence of the variable domain. In another aspect, an amino acid is placed at a position proximal to a cys residue that participates in an intrachain variable domain disulfide bond that corresponds to an amino acid found at that position in a variable domain consensus sequence subgroup that has the most sequence identity with the HVR1 and / or HVR2 amino acid sequence of the variable domain.

The present inventors succeeded in discovering specific amino acid mutations in the variable region, framework region, and constant region of TOCILIZUMAB, and this enables to reduce immunogenicity risk and the heterogeneity originated from disulfide bonds in the hinge region, as well as to improve antigen binding activity, pharmacokinetics, stability under acidic conditions, and stability in high concentration preparations.

The present invention relates to a self-molding permanent agent and a method for proceeding free-rod and free-band type permanent, more particularly to a self-molding permanent agent comprising (a) a reducing composition containing a reducing agent reducing a disulfide bond of cystine on the hair and a molding stimulant spontaneously molding to fix a hair design; (b) a molding composition inducing to mold after reacting with the molding stimulant; and (c) a softening composition releasing the action of a molding stimulant, and a method for pressing a free-rod and free-band type permanent, which overcomes a disadvantage in the conventional method for pressing a permanent that needs to wear a curling device such as rods for a permanent (perm rod) or rubber band and improves to apply a wave set without a hair-curling device for a short time, since it has a self-molding feature.

The disclosure provides proteins that can be used to determine the redox status of an environment (such as the environment within a cell or subcellular compartment). These proteins are green fluorescent protein (GFP) variants (also referred to as redox sensitive GFP (rosGFP) mutants), which have been engineered to have two cysteine amino acids near the chromophore and within disulfide bonding distance of each other. Also provided are nucleic acid molecules that encode rosGFPs, vectors containing such encoding molecules, and cells transformed therewith. The disclosure further provides methods of using the rosGFPs (and encoding molecules) to analyze the redox status of an environment, such as a cell, or a subcellular compartment within a cell. In certain embodiments, both redox status and pH are analyzed concurrently.

Compositions, kits, and methods for repairing bonds, for example, disulfide bonds, in hair or on the skin are disclosed. The compositions provide improved conditioning benefit for dry hair or moisturize the skin. The compositions also provide a long lasting moisturized feel and smooth feel to the skin or hair, without feeling greasy. The compositions contain one or more compounds that covalently crosslink at least two thiol groups in the hair or on the skin. Use of the crosslinking compositions prevents reversion of the repaired bonds to their reduced (thiol) state, for at least one week, one month, six months, or one year, after a single application of the composition. Improved methods of styling hair, for example permanent hair waving, hair curling, and hair straightening are also provided.

The invention relates to a CD47 and PD-L1 targeting bifunctional fusion protein, belongs to the field of biomedicine, and solves the problems that anti-PD-1 / PD-L1 treatment is poor in a low-immunogenicity tumor treatment effect and anti-CD47 treatment is poor in a targeting ability. The fusion protein is composed of a CD47 binding part and a PD-L1 binding part which are linked by a disulfide bond, can block the binding of CD47 and SIRPalpha, can block the binding of PD-L1 and PD-1, can achieve the effects of activating macrophages to phagocytize tumor cells and promoting antigen presentation in natural immunity, can achieve an effect of promoting the activation of tumor specific T cells in acquired immunity, and has lower blood toxicity; and as compared with the single use of anti-PD-L1 or anti-CD47 for treatment, the fusion protein has better antitumor curative effects and blood safety.

The invention relates to a method of preparing heteromultimeric polypeptides such as bispecific antibodies, bispecific immunoadhesins and antibody-immunoadhesin chimeras. The invention also relates to the heteromultimers prepared using the method. Generally, the method provides a multispecific antibody having a common light chain associated with each heteromeric polypeptide having an antibody binding domain. Additionally the method further involves introducing into the multispecific antibody a specific and complementary interaction at the interface of a first polypeptide and the interface of a second polypeptide, so as to promote heteromultimer formation and hinder homomultimer formation; and / or a free thiol-containing residue at the interface of a first polypeptide and a corresponding free thiol-containing residue in the interface of a second polypeptide, such that a non-naturally occurring disulfide bond is formed between the first and second polypeptide. The method allows for the enhanced formation of the desired heteromultimer relative to undesired heteromultimers and homomultimers.

This invention relates to a remedy for neuropathy, such as nerve injury, nerve degeneration, and hypofunction upon nerve grafting, which contains as the active ingredient galectin-1 having an amino acid sequence represented by SEQ ID NO:1 or its derivative; a protein having the amino acid sequence represented by SEQ ID NO:1 or one having a homology of 90% or more at the amino acid level with the sequence of SEQ ID NO:1 and carrying a disulfide bond(s) at least between Cys at the 16-position (Cys 16) and Cys at the 88-position (Cys 88) among cystein residues at the 2-position (Cys 2), 16-position (Cys 16), 42-position (Cys 42), 60-position (Cys 60), 88-position (Cys 88) and 130-position (Cys 130); and a process for producing the galectin-1 or its derivative protein by using an affinity column having an antibody to the above protein.

A single chain T cell receptor (scTCR) comprising an a segment constituted by a TCR α chain variable region sequence fused to the N terminus of a TCR α chain constant region extracellular sequence, a β segment constituted by a TCR β chain variable region fused to the N terminus of a TCR β chain constant region extracellular sequence, and a linker sequence linking the C terminus of the a segment to the N terminus of the β segment, or vice versa, the constant region extracellular sequences of the α and β segments being linked by a disulfide bond, the length of the linker sequence and the position of the disulfide bond being such that the variable region sequences of the α and β segments are mutually orientated substantially as in native αβ T cell receptors. Complexes of two or more such scTCRs, and use of the scTCRs in therapy and in various screening applications are also disclosed.

Described is a spray-on hydrogel comprising water-soluble PEG polymers that cross-link in situ to form a hydrogel such that the cross-links are reversible. The hydrogel can be useful as a drug delivery composition, wound dressing or surgery adjuvant. Polyethylene glycol polymer and cross-linker solutions are sprayed simultaneously through a common orifice. Cross-linking via formation of thioether or disulfide bonds is initiated upon mixing, providing rapid gelation. The hydrogel components can be derivatized with RGD peptides or analogs thereof to promote retention in / on a body compartment such as the skin, surface of the eye, or a mucosa such as the vaginal mucosa. The cross-links are reversed using a reducing solution enabling easy removal of the hydrogel by dissolution. Processes for preparation of the cross-linker, RGD derivatized PEG and RGD-linked agents are also disclosed.

Formulations, kits, and methods for rebuilding the disulfide bonds in keratin found in hair, skin, or nails. Hair that is damaged due to a hair coloring treatment and / or other reducing treatment, such as during a permanent wave, can be treated with the formulations containing one or more active agents. The formulations may be applied subsequent to a hair coloring treatment or simultaneously with a hair coloring treatment. Use of the active agent formulations during a permanent wave treatment prevents the reversion of the hair to its previous state, for at least one week, preferably at least three months, more preferably at least one year, most preferably greater than one year, after one or more than one application of the formulation. Application of the active agent formulation to skin or nails can help repair damaged disulfide bonds due to natural wear and tear or natural aging.

The present disclosure provides an effective method for the refolding of denatured proteins in solution so that properly folded, biologically active protein in solution is recovered in high yield. The refolding takes place at pressures between about 0.25 kbar to about 3.5 kbar, advantageously at about 1.5 kbar to about 3 kbar. Typically a chaotropic agent is present at a concentration which is not effective for denaturing protein at atmospheric pressure, and optionally, oxidation-reduction reagents can be incorporated in the refolding solution so that native intramolecular disulfide bonds can be formed where that is desired. The method is applicable to substantially all proteins, especially after solubilization and / or denaturation of insoluble protein aggregates, inclusion bodies, or abnormal oligomeric (soluble) aggregates.

The invention relates to an ex vivo method for the generation of a bispecific antibody, comprising the steps of: a) providing a first antibody having a first binding specificity, wherein said first antibody comprises an IgG4-like CH3 region, b) providing a second antibody having a second binding specificity which differs from said first binding specificity, wherein said second antibody comprises an IgG4-like CH3 region, c) incubating said first and second antibodies together under reducing conditions which allow the cysteines in the core hinge region to undergo disulfidebond isomerization, and d) obtaining a bispecific antibody. The invention furthermore relates to bispecific antibodies obtainable by the method of the invention.

The invention discloses a preparation method and an application of sulfhydryl / disulfide bond controllable self-crosslinked hyaluronic acid hydrogel and belongs to the field of biological materials. The hydrogel is prepared from a sulfhydryl-hyaluronic acid compound with free sulfhydryl as a raw material; by adjusting the sulfhydryl density (10-60%), the gel temperature (4-37 DEG C) and the molecular weight (0.1M, 0.3M and 1M) of hyaluronic acid, and using the oxidation-reduction transformation characteristics between sulfhydryl and disulfide bonds to control the forming time, degradation time and the mechanical properties of the gel, a controllable and injectable type intelligent hydrogel with a good three-dimensional network crosslinked structure is constructed. Meanwhile, the hydrogel of the type is single in component, avoids the introduction of exogenous toxic substances before and after gel forming, and has good biocompatibility and degradation performance. Based on the research discovery, the controllable self-crosslinked high-polymer polymer can be applied to minimally invasive repairing of tissue engineering in-situ damages and construction of an intelligent adjustable three-dimensional cell culture scaffold.

The invention relates to a chitosan modified alginate hydrogel three-dimensional porous bracket with specific in-vitro degradability and a preparation method thereof. The method comprises the following steps: dissolving sodium alga acid serving as a raw material in phosphate buffer solution; performing amidation reaction of a carboxyl group in the sodium alga acid and an amino group in a cross-linking agent cystamine or dimethyl cystinate under the activation of water-soluble carbodiimide to form a chemically crosslinked hydrogel; performing freeze drying on the hydrogel to obtain a porous bracket material of the hydrogel; and performing surface modification on the porous bracket by using chitosan. In solution of a reducing agent such as cysteine with an appropriate concentration, a disulfide bond in a hydrogel cross-bridge is degraded through a disulfide bond-sulfydryl conversion reaction, so the porous bracket is decomposed and dissolved and disappears. Therefore, the porous bracket can be used as an in-vitro cell culture template material. The hydrogel porous bracket researched by the invention has the characteristics of simple preparation, rich raw material source, low cost and availability. Various physiochemical performances, mechanical strength, degradation rate and surface properties of the bracket material are controllable within a large range.

The invention provides a disulfide-bond-introduced omega-aminotransferase mutant and application thereof. The amino acid sequence of the omega-aminotransferase mutant is disclosed as SEQ ID NO.1. The omega-aminotransferase mutant is obtained by respectively mutating 131st-site arginine and 134th-site aspartic acid of Aspergillus terreus omega-aminotransferase into cysteines. Compared with the wild type, the half-inactivation temperature of the omega-aminotransferase mutant is enhanced by 1.6 DEG C, the half life at 40 DEG C is 10.4 minutes which is prolonged by 3.5 minutes (50.7%), and the heat stability is greatly enhanced.

The present application is directed to stabilized envelope glycoprotein trimers. The trimers are stabilized by introducing disulfide bonds at certain sites in the gp41 ectodomain. DNA molecules encoding such trimers can be used to generate an immunogenic reaction.

The invention relates to a quantitative detection method for thermal-denaturation and non-denaturation bovine alpha-lactalbumin in milk and milk products by applying an enzymolysis-liquid chromatography and mass spectrometry combination technology. The quantitative detection method comprises the steps as follows: taking a certain amount of milk or milk samples, dissolving and diluting the milk or milk samples in water to obtain solution with total protein content being about 1mg / mL; after volume metering, correctly sucking 500 mu L, adding an internal standard substance, reacting disulfide bond with dithiothreitol (DTT), alkylating to protect sulfydryl produced in reaction by iodoacetamide (IAA), and then conducting constant-temperature and constant-time enzymolysis with trypsin; and separating enzymolysis products by reversed phase liquid chromatography, conducting detection with a mass spectrum multiple reaction monitoring (MRM) manner, and calculating the result by an internal standard method. The quantitation limit of the method is 0.001g / 100g; when adding amount is 0.2, 1.7 and 5.0g / 100g, the recovery rate is 98.9-110.8% (n is equal to 6) and repeatability: RSD (Relative Standard Deviation) is smaller than 7.6%; and the quantitative detection method can be applicable to the quantitative detection of samples with different contents of bovine alpha-lactalbumin.

The instant invention describes methods of separating or preferentially synthesizing dimers which are linked via at least one interchain disulfide linkage from dimers which are not linked via at least one interchain disulfide linkage from a mixture comprising the two types of polypeptide dimers. These forms can be separated from each other using hydrophobic interaction chromatography. In addition, the invention pertains to connecting peptides that result in the preferential biosynthesis of dimers that are linked via at least one interchain disulfide linkage or that are not linked via at least one interchain disulfide linkage. The invention also pertains to compositions in which a majority of the dimers are linked via at least one interchain disulfide linkage or are not linked via at least one interchain disulfide linkage. The invention still further pertains to novel binding molecules, e.g., comprising connecting peptides of the invention.

Provided are a toner for developing an electrostatic image comprising a crystalline resin having an ester bond and at least one of a sulfide bond or a disulfide bond in the main-chain, an electrostatic image developer and an image-forming process by using the same, a method of producing the toner for developing electrostatic image, and a resin particle dispersion using the same.

The invention discloses a polyurea self-repairing thermoplastic elastomer and a preparation method thereof. The preparation method comprises the following steps of introducing a monomer containing dynamic disulfide bonds into a polyurea reaction system; defoaming under the vacuum condition, curing and forming, so as to obtain the polyurea thermoplastic elastomer with self-repairing property. The polyurea self-repairing thermoplastic elastomer is prepared from the following components in parts by mass: 10 to 20 parts of diisocyanate, 45 to 70 parts of polypropylene glycol, 7 to 12 parts of chain extender, and 3 to 4 parts of catalyst. The polyurea self-repairing thermoplastic elastomer has the advantages that the self-repairing can be performed for multiple times without external conditionstimulation and special condition requirement, and the good mechanical property is realized.

The invention discloses a glucagon like peptide(GLP)-1 mutant polypeptide and a preparation method, a medicinal composition and use thereof. The mutant polypeptide is formed by bonding a Cys-containing elongation peptide to the N terminal of the glucagon like peptide-1 mutant, the mutant polypeptide itself folds to form a disulfide bond. The glucagon like peptide-1 mutant polypeptide is used for preparing a medicinal composition for treating diabetes and treating and preventing obesity. For overcoming the drawbacks of short retention time in body and need of daily injection administration of clinic GLP-1 analogue, the invention provides the GLP-1 analogue which has a longer half-life period. With the longer half-life period, the GLP-1 mutant polypeptide is not required to be injected into a patient every day, and can increase the obedience of the patient effectively.

The present invention relates to an isolated Clostridial neurotoxin propeptide having a light chain region, a heavy chain region, where the light and heavy chain regions are linked by a disulfide bond, and an intermediate region connecting the light and heavy chain regions. An isolated nucleic acid molecule encoding a Clostridial neurotoxin propeptide is also disclosed. Also disclosed is an isolated, physiologically active Clostridial neurotoxin produced by cleaving a Clostridial neurotoxin propeptide, a vaccine or antidote thereof, and methods of immunizing against or treating for toxic effects of Clostridial neurotoxins. Methods of expressing recombinant physiologically active Clostridial neurotoxins are also disclosed. Also disclosed is a chimeric protein having a heavy chain region of a Clostridial neurotoxin and a protein with therapeutic functionality. A treatment method is also disclosed.

The invention discloses a preparation method and application of a PD-1 / CTLA-4 (programmed death-1 / cytotoxic T lymphocyte antigen-4) bispecific antibody. The preparation method comprises the following steps of using a single-chain antibody gene as a template and introducing disulfide bond into a variable region of the antibody by a particular high-efficiency amplification primer through PCR (polymerase chain reaction) and Overlap PCR through a gene engineering technique, so as to prepare a PD-1 / CTLA-4 bispecific antibody fragment; cloning the gene fragment to a chronic virus expression carrier, and transfecting 293T cells to package the virus; transducing a CIK (cytokine induced killer) cell, separating PBMC (peripheral blood mononuclear cell) from peripheral blood of a health person, using CD3 / CD28 magnetic beads to stimulate the growth of the T cell, and co-culturing the chronic virus and the T cell, so as to obtain a novel anti-tumor T cell which can block an immunosuppression signal of the tumor and tumor micro environment source, and enhance the function of killing cancer cells.