83 results about "Disulfide bond reduction" patented technology

The invention concerns methods and means for preventing the reduction of disulfide bonds during the recombinant production of disulfide-containing polypeptides. In particular, the invention concerns the prevention of disulfide bond reduction during harvesting of disulfide-containing polypeptides, including antibodies, from recombinant host cell cultures.

The invention relates to a quantum dot fluorescent probe and application thereof. The invention is characterized in that the quantum dot fluorescent probe utilizes a difunctional cross-linking agent long-chain succinimidyl-4-[N-maieimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate] (SMCC) to undergo a cross-linking reaction with an amino group on the surface of an amino quantum dot; a maleimide group is produced on the surface of the quantum dot; a to-be-labeled antibody is reduced by a reducing agent dithiothreitol (DTT) so as to reduce a disulfide bond into a mercapto group; and the mercapto group and the maleimide group form a covalent bond so as to realize labeling of the quantum dot with the antibody. The quantum dot fluorescent probe is applied to (1) detection of one protein oncofetal antigen or (2) detection of a plurality of protein CEA, euron-specific enolase (NSA) and a cytokerain fragment 19CYFRA21-1, wherein the detection limit of detection (1) is 38 pg / ml, and the detection limit of detection (2) is 0.9 ng / ml.

The invention relates to an extraction kit, an extraction method and a detection method for drugs in hair. The extraction kit comprises a composite extract containing a disulfide bond reducing agent,a hydrogen bond blocker, a hair expansion agent and a solvent, wherein the composite extract is alkaline and has a pH of 8.5 to 10. The extraction kit for drugs in hair hydrolyzes the hair by using areduction method with no need of special pulverizing equipment and extraction equipment during the pretreatment and with short hydrolysis time and simple operation of the whole extraction process, andthe reduction method is more suitable for the requirements of rapid detection of laboratory forensic identification compared with the conventional methods such as acid methods, alkali methods, methanol solvent methods, buffer salt solution methods and enzymatic methods. Furthermore, the reagents used in the composite extract in the extraction kit are all water-soluble reagents, which are easy tobe extracted by the organic reagent liquid, so that the solution to be detected can be quickly separated. And the reagents used are basically pollution-free to the environment, non-toxic to the operator, easily removed without residue, and basically damage-free to instruments and equipment.

The invention discloses an 18F-labeled compound and a pod protease targeted PET imaging probe, which relates to the technical fields of radiopharmaceuticals and nuclear medicine. The invention provides a compound shown as a formula (I), asparagine site shearing and disulfide bond reduction are carried out in a tumor microenvironment with high expression of pod protease, and a radioactive dimer 18F-1-dimer is formed by virtue of a biocompatible CBT-Cys click condensation reaction, so that the tumor imaging effect is improved. The compound with a structure shown in the formula (I) is synthesizedby adopting a one-step ion exchange 18F labeling method, the method is simple to operate, and preparative HPLC for further purification is not needed. The invention further provides the pod proteasetargeted PET imaging probe which is a compound shown in (I). The PET imaging probe has the advantages of high stability, high sensitivity, strong specificity, good safety and the like.

The invention belongs to the technical field of biological medicine, and discloses protein combined nanometer selenium, and a preparation method and applications thereof. According to the preparationmethod, under the reducing action of a disulfide bond reducing agent, the internal space structure of a disulfide bond containing protein or a disulfide bond containing polypeptide is opened so as toobtain mercapto groups with reducing activity, and a part of the mercapto groups are used for converting selenium compounds into elemental selenium through reducing, the rest mercapto groups are usedfor forming intramolecular and intermolecular disulfide bonds, so that self assembling of protein combined nanometer selenium is obtained; the preparation method is capable of improving the dispersion performance and the stability of nanometer elemental selenium in solution greatly. It is confirmed by animal experiments that the active red elemental selenium is high in bioavailability and low intoxicity. The protein combined nanometer elemental selenium possesses the functions of protein and nanometer selenium, is small in nanometer particle size, is narrow in distribution range, can be stored in the state of liquid phase, is convenient for oral administration, injection, or spraying.

The invention relates to a double-sensitive amphiphilic copolymer containing a Schiff base and a mercapto group on a same side group, as well as a preparation method and application of the double-sensitive amphiphilic copolymer. The copolymer is the amphiphilic copolymer which introduces an acid-sensitive bond, namely the Schiff base and a redox-sensitive group, namely the mercapto group on the same side group on the basis of a polyethylene glycol / polyester amphiphilic copolymer containing a side carboxyl group in a polyester segment. The copolymer can form acid-sensitive and redox-sensitive reversible cross-linked nanoparticles with physical entrapment of a medicament by self-assembly, namely that a disulfide bond is formed by oxidation of the mercapto group for cross-linking, and the fracture of the mercapto group or the Schiff base is produced by reduction of the disulfide bond, thereby disintegrating a cross-linked structure. The fracture of any bond of the Schiff base and the disulfide bond can promote the disintegration of the double-sensitive polymer nanoparticles and the release of the medicament and further improve the bioavailability of the medicament in an acidic micro-environment of tumor cells and high concentration of glutathione (GSH).

The invention discloses a formaldehyde-free and strength loss-free crease-resistant finishing method for cotton fabrics, and belongs to the field of crease-resistant finishing of textiles. A lipoic acid analogue containing disulfide bonds (or sulfydryl) and carboxyl simultaneously is used as a crease-resistant finishing agent for cotton fabrics, and a formaldehyde-free and strength loss-free crease-resistant finishing method for cotton fabrics is established. The method comprises the steps of firstly dipping scoured and bleached cotton fabrics in a dimethyl sulfoxide solution of the lipoic acid analogue, and completing an esterification reaction under the action of a dehydrating agent and a catalyst; and reducing a disulfide bond in the lipoic acid analogue into two sulfydryl groups by using a reducing agent, and finally oxidizing the sulfydryl groups into a disulfide bond under a certain tension to form a covalent cross-linked structure so as to obtain the durable anti-wrinkle cottonfabrics. The finished cotton fabrics are strong, free of damage, excellent in crease resistance and good in washable stability. The molecular structure of the lipoic acid analogue does not contain formaldehyde, so that the problems of formaldehyde release and the like do not exist in the processing process or taking process, and the safety of the anti-wrinkle cotton fabrics is ensured.

The invention provides a method for extracting a poultry feather keratin by using water insoluble ionic liquid, comprising the follow steps: 1) washing, drying and crushing a poultry feather; 2) adding a water insoluble ionic liquid with stirring for 3-6h at the temperature of 70-100 DEG C; and 3) after the reaction, adding water, carrying out centrifugation to form a three-phase system of water insoluble ionic liquid / undissolved poultry feather / water, so as to obtain a crude keratin solution. In a preferred step 2), a disulfide bond reducing agent is added, and a little water is added to dissolve the disulfide bond reducing agent. The invention also provides a poultry feather keratin solution extracted by the method. The method for extracting the poultry feather keratin by using the water insoluble ionic liquid, has the advantage of simple separation of keratin, is convenient recovery of ionic liquid and friendly to environmental protection, and can be widely applied.

Compositions and methods for minimizing antibody disulfide bond reduction are described. In one aspect, a composition is provided for culturing mammalian host cells to express an antibody including an anti-reduction agent that minimizes reduction of a disulfide bond in the antibody or fragment thereof. In some other aspects, methods for minimizing disulfide bond reduction; increasing production of an antibody or fragment thereof with intact native disulfide bonds; increasing a ratio of non-reduced to reduced antibody or fragment thereof; producing a therapeutic antibody or fragment thereof by adding a sufficient amount of an anti-reduction agent to a cell culture media, pre-harvest cell culture fluid, or harvest cell culture fluid are described. In another aspect, minimizing disulfide bond reduction in an antibody or fragment thereof culturing the host cell in a concentration of at least about 20% O2 is described.

The invention discloses a reagent and a method for improving reverse transcriptase performance. The main component of the reagent is a mercapto sealing agent, or a disulfide-bond reducing agent, or a combination of the two. The invention also discloses a method for improving reverse transcriptase performance. According to the method, the mercapto sealing agent or the disulfide-bond reducing agent or the composition of the two is introduced into the reverse transcriptase. With the reagent provided by the invention, reverse transcriptase performance can be effectively improved, such that the reverse transcriptase has higher extension capacity and higher reaction sensitivity, and higher reverse transcription temperature can be adopted. Therefore, reverse transcription difficulty caused by template RNA secondary structure is solved. With the reagent and the method, high-ratio full-length cDNA library can be constructed, and relatively long cDNA can be synthesized.

The invention belongs to the technical field of biological medicine, and discloses a novel albumin-siRNA composite nanometer particle, and a preparation method and applications thereof. According to the preparation, the internal space structure of albumin is destroyed using a disulfide bond reducing agent, and proteins containing mercapto group active groups are obtained, siRNA and / or a modified substance SH-siRNA is added, and a selenium compound is added so as to obtain the novel albumin-siRNA composite nanometer particle. The preparation method is simple; the nanometer size is uniform at different dilution concentrations, the dispersion performance is excellent, the stability in artificial gastric juice, intestinal juice, and blood environment is excellent; and in addition, the novel albumin-siRNA composite nanometer particle possesses intracellular reduced glutathione microenvironment responsiveness; the novel albumin-siRNA composite nanometer particle possesses oral administrationand injection administration living tumor targeting characteristic, is capable of increasing siRNA anti-cancer drug oral administration or injection administration anti-cancer activity, and reducingtoxic or side effect.

The invention provides an ultraviolet-resistant moisture absorbing and heating protein tencel fiber and a preparation method thereof. The preparation method comprises the steps: dissolving cellulose pulp into an N-methylmorpholine-N-oxide aqueous solution, and carrying out modification by using a sulfhydryl compound under the action of a molecular sieve based catalyst to obtain a modified cellulose spinning mother solution; carrying out enzymolysis on wool in water by using a protein-disulfide reductase and a solid protease to obtain a keratin solution; and mixing the keratin solution and themodified cellulose spinning mother solution, and then, preparing the ultraviolet-resistant moisture absorbing and heating protein tencel fiber by using a dry-jet wet spinning method. The protein tencel fiber prepared by using the preparation method has relatively high protein content and has ultraviolet-resistant moisture absorbing and spontaneous heating functions, and both the wet breaking strength and dry breaking strength of the fiber are improved.

The invention belongs to the field of degradable medical polymer synthesis and design and preparation of anti-cancer drug conjugates, and discloses adriamycin polyprodrug nano-micelle with reducibility response and a preparation method and application thereof. Hydroxyethyl methacrylate and 4, 4'-dithiodibutyric acid are adopted as basic raw materials, amino of adriamycin is modified, disulfide bond primitives are introduced, an adriamycin prodrug monomer DOXM with disulfide bond reducibility response is prepared, and polyprodrug amphiphilic molecules PDMA-b-PDOXM are prepared through RAFT. ThePDMA-b-PDOXM has very high drug loading capacity, and the water solubility and the stability of the drug are improved. The disulfide bonds enable the drug carrier to respond to a tumor cell reductivemicroenvironment after reaching a tumor targeting site to release a small molecular raw drug adriamycin, so that the drug toxicity is reduced and an anti-cancer drug can be intelligently released intumor cells.

The invention discloses sulfhydrylated hyaluronic acid as well as a preparation method and application thereof. The sulfhydrylated hyaluronic acid is obtained by sulfhydrylation modification of hydroxyl sites of hyaluronic acid or salt thereof. According to the invention, the hyaluronic acid or salt thereof reacts with a disulfide compound to generate a hyaluronic acid disulfide compound, and then a reducing agent is added to reduce disulfide bonds into a sulfydryl group, so the sulfhydrylated hyaluronic acid is obtained. The sulfhydrylated hyaluronic acid has the characteristics of controllable sulfydryl substitution degree, fast dissolution, fast gelling and good biocompatibility, and can be applied to the fields of skin care products, joint injection, tissue filling, cell culture and the like.

The invention discloses a positioning method of a polypeptide disulfide bond. The comprises the steps that to-be-tested polypeptide is partially reduced in the solution environment of a disulfide bond containing reducing agent; the partially reduced polypeptide is alkylated in the solution environment of a polypeptide containing denaturing agent and an alkylated reagent; the alkylated polypeptide is completely reduced in the solution environment of the polypeptide containing denaturing agent and the alkylated reagent; a matrix auxiliary laser desorption ionization-flight time mass spectrum is used for performing molecular weight on products; samples qualified through detection are subjected to hygroplasm combination tandem mass spectrum detection, and mass spectrometric data is obtained; the spectrometric data is converted into a data format compatible with the mass spectrum identification software, then, the mass spectrum identification software is used for performing search comparison on the spectrum data and a reference polypeptide sequence database, and the proportion of the connecting position of the polypeptide disulfide bond and different connection modes is calculated. The method is small in sample amount requirement, low in purity requirement, easy to implement, high in repeatability, small in analysis difficulty, high in result accuracy, high in flux and low in cost.

The invention discloses a preparation method of an As2O3 albumin nano medicine. The preparation method includes the following steps: 1) dissolving As2O3 powder in a NaOH solution and regulating the pH with diluted hydrochloric acid to prepare an As2O3 solution for drug supporting of albumin in later use, dissolving human serum albumin marked by folic acid molecules in a Dulbecco's phosphate buffer liquid without calcium and magnesium ions to obtain a human serum albumin solution, and adding a disulfide bond reducing agent to the human serum albumin solution and performing a reaction in a water bath pot with gentle stirring to obtain the albumin solution treated by the disulfide bond reducing agent; 2) adding the As2O3 solution to the albumin solution treated by the disulfide bond reducing agent, and stirring the mixture at room temperature to obtain an As2O3 albumin mixed solution; and 3) dialyzing the As2O3 albumin mixed solution in a dialysis bag at low temperature with a D-PBS solution to remove residual As2O3, disulfide bond reducing agent and side products thereof to obtain the As2O3 albumin nano medicine.

The present invention relates to a qualitative and quantitative method for a thio thiolation post-translational modification peptide segment. The method is divided into two routes such as a route A and a route B, wherein the route A comprises that a peptide segment produced through proteolysis and iodoacetamide beads are incubated, the mercapto peptide segment and the thio mercapto peptide segmentare enriched, the disulfide bond of the thio mercapto peptide segment is reduced and released with a reducing agent, the released thio mercapto peptide segment can be labeled with a cleavable isotopeaffinity tag, and finally the linking arm of the cleavable isotope affinity tag is cleaved with a lysis liquid, and the route B comprises that protein containing mercapto and thio mercapto is labeledwith a cleavable isotope affinity tag, proteolysis is performed, the mercapto peptide segment and the thio mercapto peptide segment are subjected to affinity enrichment, and finally the linking arm of the cleavable isotope affinity tag linked to the mercapto peptide segment and the thio mercapto peptide segment is cleaved with a lysis liquid. According to the present invention, the qualitative and quantitative method has advantages of good selectivity, low false positive and the like, and can achieve the qualitation and the quantification of the scaled thio thiolation peptide segment in the complex proteome sample.

The invention discloses a preparation method of surface plasma resonance chips. The preparation method is characterized in that a disulfide bond reducing agent is adopted to modify biological protein molecules containing hydrosulfide group and / or disulfide bond; then, protein substrate is established on the surface of a chip by means of metal surface molecule self-assembly technology; and further modification is carried out to realize the preparation of a novel sensing chip used in SPR biosensor. The preparation method has the advantages of low preparation cost, simple operation, less time consumption and safety and harmlessness; moreover, the prepared chip has the functions of commercialized CM5 chips, thereby providing new possibilities for solving the prior problems of fewer types of surface plasma resonance chips and high cost.

The present disclosure relates to the use of host cell protein biomarkers to assess disulfide bond reduction in compositions comprising a protein of interest. In some embodiments, the disclosure relates to methods of predicting the occurrence of disulfide bond reduction or low molecular weight protein species in compositions comprising a protein of interest, wherein the expression or activity level of at least one host cell protein is measured and provides a benchmark value associated with the occurrence of disulfide bond reduction or low molecular weight species of said protein of interest. In some embodiments, the disclosure relates to methods of producing a protein of interest, wherein host cells capable of producing the protein of interest are cultured, the expression or activity level of at least one host cell protein is measured, and downstream isolation of the protein of interest is informed by the host cell protein measurements.

The invention discloses a preparation method of a plant protein derivative with physiological activity and a nutrition supplementing function and belongs to the field of plant protein deep processing. According to the preparation method disclosed by the invention, the plant protein is used as a raw material and subjected to the processes of enzyme hydrolysis, clostridium thermoaceticum, biochemical separation and the like, and the product is a plant protein enzymatic hydrolysate with cysteine residue peptide as the important ingredient. According to the preparation method disclosed by the invention, abundant plant protein resource containing cysteine is used and subjected to enzymatic hydrolysis to obtain the plant protein derivative with the physiological activity and the nutrition supplementing function, and therefore the preparation method is of great significance.

The invention discloses a method for preparing a lysozyme composite food packaging film. The method comprises the following steps: 1, respectively preparing a neutral buffer solution of lysozyme and adisulfide bond reducing agent tris(2-carboxyethyl)phosphorus hydrochloride solution, mixing the two solutions to be uniform so as to prepare a composite solution; 2, taking out one part of the composite solution in the step 1 and adding into a packaging bag, enabling the inner wall of the packaging bag to be completely infiltrated by the composite solution, standing, and pouring the composite solution in the packaging bag; 3, drying the packaging bag treated by the step 3, and coating a protein film layer on the inner wall of the packaging bag; 4, repeating the step 2 and step 3, superposingthe protein films layer by layer, and forming the lysozyme composite food packaging film on the surface of the packaging bag. According to the preparation method disclosed by the invention, the surface medium of an ordinary packaging material is changed, the packaging material is attached to the surface of a general package by virtue of adhesion of the proteins, the dense film can be rapidly formed, and the application effect of the packaging film in food preservation is improved.

The invention relates to a method for improving surface bioactivity and osseointegration performance of a polyether-ether-ketone base material, which sequentially comprises the following steps: (1) soaking the polyether-ether-ketone base material in a mixed solution of a protein solution and a disulfide bond reducing agent to obtain a nano-film modified polyether-ether-ketone base material; and (2) soaking the nano-film modified polyether-ether-ketone base material obtained in the step (1) in a calcium-phosphorus solution, and performing closed incubation to obtain the hydroxyapatite coating modified polyether-ether-ketone base material. According to the method for improving the surface biological activity and bone integration performance of the PEEK base material, the problem that in the prior art, induced mineralization cannot be conducted on the surface of the PEEK base material is solved, and the modified PEEK base material can serve as a tissue repair material to induce formation of new tissues and mineralization of bones; the material is expected to become a novel hard tissue substitute material.

The invention discloses a making method and usage of artificial feather-root neurobridge-pipe and neurobridge-filament, which is characterized by the following: cleaning the feather-root; degreasing; disposing feather-root protein to change its structure by the disulfide bond reducer, acid, polypeptide chain non-covalent bond denaturant and alkali; the bulk factor of feather-root and the each relating reagent weight is 5%-20%; the adaptation density of reagent is (0.1-1.0)mol / L; the temperature and time of each processing procedure is 20 Deg C.-40Deg C. and 5-30 hours separately; adapting frozen section method to cut the disposal feather-root into the filament with the diameter of 20mu m-100mu m; formatting the bridge-filament. The method provides a transient succedaneum of injured peripheral nerve of human reconditioning arrangement for medical traumatology department.

The invention provides a detection reagent and a detection method for detecting the content of sodium polydithiodipropane sulfonate in an acid copper electroplating solution. The used detection reagent comprises a reducing agent, a buffer solution and a color developing agent, and the reducing agent is a reducing reagent for reducing disulfide bonds in the sodium polydithiodipropane sulfonate intosulfydryl. The buffer solution is a buffer solution with the pH value of 8-10. The color developing agent is 5,5'-dithiobis (2-nitrobenzoic acid). The method has the advantages of simple operation, rapidness, convenience, good repeatability and high accuracy. The relative error is about 0.8%-4%, the detection limit is as low as 2[mu]mol / L, and the detection reagent is suitable for real-time monitoring of plating solution components of acid copper electroplating factories and electrolytic copper foil enterprises and stabilizes the product quality.

Disclosed are methods for testing the presence and / or activity of glutathione system components and thioredoxin system components during the manufacturing process of disulfide bond-containing proteins. Also disclosed are methods for mitigating reduction of disulfide bonds during the manufacturing process, and for lowering the reduction potential of disulfide bond- containing proteins. Provided arecompositions, kits, and methods for mitigating reduction and diminishing reduction potential of disulfide bond-containing proteins during protein manufacturing processes.

The invention relates to a zwitterionic cross-linking agent, a zwitterionic polymer and a zwitterionic injectable hydrogel for preventing postoperative adhesion, and a preparation method of the cross-linking agent, the polymer and the hydrogel. The method comprises the following steps of: subjecting N-methyl diethanolamine to acylation and ring-opening addition so as to synthesize the zwitterioniccross-linking agent with double bonds at two ends; subjecting a zwitterionic compound monomer and a disulfide bond monomer to free radical polymerization and disulfide bond reduction reaction so as to obtain the zwitterionic polymer with a sulfhydryl group in a side chain; and then respectively dissolving the cross-linking agent and the polymer in a phosphate buffer solution, and performing 'thiol-ene' addition reaction to obtain the zwitterionic injectable hydrogel. The prepared zwitterionic cross-linking agent and the zwitterionic polymer have excellent anti-protein performance, and the corresponding zwitterionic injectable hydrogel formed by the zwitterionic cross-linking agent and the zwitterionic polymer has better biocompatibility and anti-cell adhesion, and can be used as an anti-adhesion material for preventing postoperative adhesion.

The invention relates to the technical field of biology, in particular to a glycoprotein N-sugar chain analysis method. The method comprises the following steps: a) carrying out protein expansion denaturation and disulfide bond reduction treatment on glycoprotein to be analyzed by using 2-methylpyrimidine borane and a disulfide bond reducing agent; b) carrying out alkylation treatment on the protein treated in the step a), and carrying out enzyme digestion on N-glycosidase F; and c) drying the sample subjected to enzyme digestion, marking the dried sample with a marker, and analyzing the N-carbohydrate chain according to the marker, wherein the marker is procaine amide and / or a salt thereof. The method can obviously shorten the operation process and increase the detection flux, and is more environment-friendly and easy to use.

Compositions and methods for minimizing antibody disulfide bond reduction are described. In one aspect, a composition is provided for culturing mammalian host cells to express an antibody including an anti-reduction agent that minimizes reduction of a disulfide bond in the antibody or fragment thereof. In some other aspects, methods for minimizing disulfide bond reduction; increasing production of an antibody or fragment thereof with intact native disulfide bonds; increasing a ratio of non-reduced to reduced antibody or fragment thereof; producing a therapeutic antibody or fragment thereof by adding a sufficient amount of an anti-reduction agent to a cell culture media, pre-harvest cell culture fluid, or harvest cell culture fluid are described. In another aspect, minimizing disulfide bond reduction in an antibody or fragment thereof culturing the host cell in a concentration of at least about 20% O2 is described.

The invention provides a nano-platform for overcoming the vector epitope inhibition effect of a vaccine. Albumins used as a raw material are reduced, thermopolymerizied, oxidized and concentrated, andthen are externally linked with an antigen epitope to obtain a target vaccine vector, wherein the reduction is carried out to reduce disulfide bonds in the albumins into sulfhydryl groups; the thermopolymerization is carried out to obtain nano-particles formed in an albumin self-assembling manner; and the oxidation is carried out to oxidize the sulfhydryl groups into the disulfide bonds. The nano-platform is a nano-vaccine vector having the advantages of completeness in inhibiting the production of vector protein antibodies, green and simple synthesis process, no involving of toxic chemicalsor complicated processes, low cost, small batch difference and great clinical transformation prospect.