59 results about "Protein disulfide-isomerase" patented technology

The present disclosure describes methods and systems for improving the expression of a properly folded, biologically active protein of interest in a cell free synthesis system. The methods and systems use a bacterial cell free extract having an active oxidative phosphorylation system, and include an exogenous protein chaperone. The exogenous protein chaperone can be expressed by the bacteria used to prepare the cell free extract. The exogenous protein chaperone can be a protein disulfide isomerase and / or a peptidyl-prolyl cis-trans isomerase. The inventors discovered that the combination of a protein disulfide isomerase and a peptidyl-prolyl cis-trans isomerase produces a synergistic increase in the amount of properly folded, biologically active protein of interest.

The invention discloses a method for preparing oligomeric fucosylated glycosaminoglycan which is prepared by depolymerizing fucosylated glycosaminoglycan by a depolymerization method of peroxide catalyzed by a 4th period transition metal ion in an aqueous medium, and the preparation method has mild reaction condition, good reproducibility and stability, high pyrolysis selectivity and uniform and controllable product quality. The polysaccharide molecule number of the obtained oligomeric fucosylated glycosaminoglycan using GalNAc as a reducing end is not less than 80 percent, the weight average molecular weight is about 6, 000-20, 000Da, and the protein disulfide isomerase (PDI) is 1.0-2.0.

A method of treating an infectious disease caused by a pathogen comprising administering to a subject in need thereof an effective amount of one or more compounds that inhibit the function of a molecular chaperone, wherein said compound is other than tizoxanide or nitazoxanide.

The invention discloses an E.tenella protein disulfide linkage isomerase gene EtPDI (Clone ID is BW1-E06,and the Genbank accession number of is EF552214). The gene is connected with a procaryon expression vector pGEX-4T-2; a procaryon expression recombination plasmid pGEX-4T-EtPDI is constructed and is expressed in a colibacillus system; and most of the expressed recombining protein exists in a soluble form. The recombining protein 4T-EtPDI is purified to carry out SPS-PAGE and is transferred to a PVDF film; and antiserum of E.tenella oocyst oral immunized chicken is used as first resistance and goat anti-chicken IgG is used as second resistance to carry out Western-blot analysis, thereby indicating that the gene has certain antigen. The gene is used for preparing an anti-chicken coccidiosis drug and an anti-chicken coccidiosis vaccine.

The invention discloses a method for preparing oligomeric fucosylated glycosaminoglycan which is prepared by depolymerizing fucosylated glycosaminoglycan by a depolymerization method of peroxide catalyzed by a 4th period transition metal ion in an aqueous medium, and the preparation method has mild reaction condition, good reproducibility and stability, high pyrolysis selectivity and uniform and controllable product quality. The polysaccharide molecule number of the obtained oligomeric fucosylated glycosaminoglycan using GalNAc as a reducing end is not less than 80 percent, the weight averagemolecular weight is about 6, 000-20, 000Da, and the protein disulfide isomerase (PDI) is 1.0-2.0.

The present invention relates to a method for mass production of a recombinant protein comprising the step of culturing a yeast transformed with: a recombinant gene construct comprising a yeast promoter, a gene coding a signal sequence and a gene coding a target protein; and also with one or more genes coding folding accessory protein selected from the group consisting of PDI1 (protein disulfide isomerase 1), SEC23 (secretory 23), TRX2 (thioredoxin 2) AHA1 (activator of heat shock protein 90 ATPase), and SCJ1 (S. cerevisiae DnaJ) followed by culturing the transformed yeast.

The invention relates to a method for performing fusion expression on a protein containing a disulfide bond, in particular to a method for performing fusion expression on a recombinant protein, in particular a eukaryotic recombinant protein of which the activity is closely related with the forming of the (intrachain and interchain) disulfide bond in an eukaryotic host cell. An exogenous recombinant protein is expressed in a pichia pastoris expression system by a method for fusing human protein disulphide isomerase (hPDI) or a mutant of the hPDI. The method is suitable for expressing a eukaryotic exogenous protein of which protein activity or protein folding (comprising forms of a monomer, a dimmer or a polymer) are resistant to the disulphide bond. The protein expressed by the mode can be secreted out of a cell easily, and is high in yield, products cannot be gathered on a large scale and can be captured and purified easily, and the obtained target protein is uniform in N tail end, and high in activity. The recombinant protein expressed by the method can be industrially produced on a large scale.

The invention discloses a lactase mutator with optimized codon and high specific activity and a secretory expression method thereof. The lactase gene cloned in bifidobacterium animalis is optimized for codon of the gene and the GC content on condition that the amino acid sequence of the gene is not changed according to the preference of the codon of Pichia pastoris; the optimized lactase gene is shown as SEQ ID NO.2.After optimizing, 461 basic groups are changed; the percentage of GC% is lowered to 53.79% from 61.11%t; the enzymatic activity of the lactase is obviously increased after the codon is optimized. Moreover, the method shows that: since the lactase gene and protein disulfide isomerase are transformed into the Pichia pastoris cell, the relative activity of the lactase is obviously increased; and the secretory expression content of the lactase gene in the Pichia pastoris is obviously increased.

The invention discloses an expression system of fusion protein from human serum albumin and interleukin-1 receptor antagonist. The expression system comprises a host cell and an expression vector transferred into the same. The expression vector comprises a first expression vector with an inserted fusion protein gene and a second expression vector with an inserted protein disulfide isomerase gene.The fusion protein gene comprises the human serum albumin and the interleukin-1 receptor antagonist. The host cell is Pichia pastoris GS115. The co-expression host of PDI (protein disulfide isomerase) and IGH (immunoglobulin heavy) is established, and expression index of the IGH is evidently increased. The PDI is expressed intracellularly, the IGH secretory expression is subjected to extracellular secretory expression, and accordingly no newly generated other proteins occur when concentration of the IGH in collected medium supernate increases.

The present disclosure describes methods and systems for improving the expression of a properly folded, biologically active protein of interest in a cell free synthesis system. The methods and systems use a bacterial cell free extract having an active oxidative phosphorylation system, and include an exogenous protein chaperone. The exogenous protein chaperone can be expressed by the bacteria used to prepare the cell free extract. The exogenous protein chaperone can be a protein disulfide isomerase and / or a peptidyl-prolyl cis-trans isomerase. The inventors discovered that the combination of a protein disulfide isomerase and a peptidyl-prolyl cis-trans isomerase produces a synergistic increase in the amount of properly folded, biologically active protein of interest.

The invention provides compounds of formula (I) that inhibit PDI, for use in methods to treat or prevent a disease or condition in a subject that would benefit by inhibition of PDI. Formula (I)

The invention relates to a preparation method of a recombinant protein in the field of gene engineering and in particular relates to a preparation method of a recombinant human acidic fibroblast growth factor (haFGF) protein. The preparation method comprises the following steps: (1) designing primers according to the full-length sequence of the haFGF to obtain an original gene segment through cloning, and replacing a codon which exists in the original gene segment and is unfavourable to be expressed by escherichia coli, thus obtaining an optimized haFGF gene, wherein the nucleotide sequence of the optimized haFGF gene is shown in SEQ ID NO.1; cloning to obtain an original gene segment of a molecular chaperone PDI (protein disulfide isomerase), and replacing a codon which exists in the original gene segment and is unfavourable to be expressed by escherichia coli, thus obtaining an optimized PDI gene, wherein the nucleotide sequence of the optimized PDI gene is shown in SEQ ID NO.2; (2) linking and cloning the optimized haFGF gene, the optimized PDI gene, tag sequences and a protease cutting site sequence into a vector to obtain an efficient expression vector through construction; or firstly constructing standby vectors of part of the sequences, and then linking the remaining sequences on part of the sequences.

The present invention provides a method for treating or ameliorating the effects of a neurodegenerative disorder in a subject in need thereof. The method includes, for example, administering to the subject an effective amount of a compound selected from:combinations thereof, or an N-oxide, crystalline form, hydrate thereof, or a pharmaceutically acceptable salt thereof. The present invention also provides a method for treating or ameliorating the effects of a condition associated with increased protein disulfide isomerase (PDI) activity and a method of modulating PDI activity in a cell. The present invention also provides compounds, salts, compositions and kits useful for the provided methods.

Protein disulfide isomerase inhibitors according to formula Iare described, wherein R1 is an aryl or cycloalkyl group, R2 is selected from the group consisting of CN, SO2CH3, NO2, CO2R3, CONHR3, NMe2, and CF3, R3 is selected from H or lower alkyl, R4 is H, halogen, CN, or COOH, and X and Y are C—H or heteroatoms selected from the group consisting of N, O, and S. The protein disulfide isomerase inhibitors can be used for the treatment of cancer in a subject.

The invention relates to a method for preparing a thrombolytic medicament Reteplase (rPA) directly through an escherichia coli expression system without inclusion-body renaturation. A gene segment of the rPA is obtained through PCR amplification, and is cloned into a vector pET40b to construct a pET40b-rPA recombinant plasmid, and the rPA is made to perform fusion expression with the protein disulfide isomerase DsbC in the vector pET40b. The recombinant plasmid is transformed into the escherichia coli BL21 (DE3), and induction expression conditions of IPTG and lactose are respectively established optimally. The obtained target fusion protein is mainly expressed by soluble components, and is purified by Ni-NTA affinity chromatography and processed by a Xa factor (or formic acid or thrombin) to form the high-purity rPA target protein. Fibrin plate method detection results show that the fusion protein DsbC-rPA obtained by the method and the rPA protein obtained by removing a label and purifying have obvious thrombolytic activity.

The invention discloses a process method for synthesizing a lactic acid-lysine copolymer by catalytically opening a loop and copolymerizing with acetic bicyclo-guanidine (the molar content of diaminocaproic acid is 1-5 percent). The lactic acid-lysine copolymer is synthesized by taking acetic bicyclo-guanidine as a catalyst, taking lactide and lysine morpholine diketone as monomers, performing body loop opening copolymerization and undergoing a phenmethyl carboxide removing action. The process method has characteristics that: the acetic bicyclo-guanidine serving as a catalyst is bionic organic guanidinium which is effective, nontoxic and free from metals; the monomer transformation ratio is high (more than or equal to 95 percent); the yield is over 93 percent; a product does not contain any metal or other toxic residue, and has high biological safety; the number average molecular weight is adjustable in the range of 1.5-2.8*10<4>, and the molecular weight distribution is narrow (PDI (Protein Disulfide Isomerase) is less than or equal to 1.30); the molar content of lysine in the copolymer is adjustable in the range of 1-5 percent; and the synthesized lactic acid-lysine copolymer isan amphiphilic functional group biodegradable polymer, is suitable for serving as targeted and controlled release medicament carriers, and can be applied on other aspects in the field of biological medical tissue engineering.

The invention discloses a method for increasing expression amount of pichia pastoris for secreting and expressing plectasin. The method is characterized in that a pichia pastoris genome is used as a template, a PDI (protein disulfide isomerase) gene is amplified, a PDI segment is treated by BstBI and EcoRI double enzyme digestion, and is connected to a pPIC6aphlaA carrier segment which is also treated by the BstBI and EcoRI double enzyme digestion, and an expression carrier pPIC-PDI is formed; the PDI is expressed in a plectasin expression strain, three pairs of disulfide bonds of the plectasin are promoted to form a correct space structure, the plectasin is smoothly secreted and expressed out of cells, and the purpose of increasing the expression amount of plectasin is realized. The method has the advantage that the expression amount of plectasin is increased by 30%.

The invention verifies that protein disulfide isomerase A3 expression in the cancer tissues and adjacent tissues of hepatocellular carcinoma is different through screening the protein having differential expression in the cancer tissues and adjacent tissues of hepatocellular carcinoma and carrying out western blot experiment. The invention discloses the application of the protein disulfide isomerase A3 to the detection of liver cancer and / or susceptibility of liver cancer and the application of the antibody of the protein disulfide isomerase A3 to the detection of liver cancer and / or susceptibility of liver cancer. Therefore, as the potential marker of liver cancer, the protein disulfide isomerase A3 can serve as a marker of the prognosis molecule of liver cancer and a target molecule of clinical treatment.

The present invention relates to a method for producing a recombinant protein capable of increasing expression rate of a target protein and also improving solubility and folding of the expressed target protein using a modified protein disulfide isomerase (PDI) as a fusion partner, and an expression vector containing the modified PDI gene as a fusion partner. The method for preparing a recombinant protein using a modified PDI as a fusion partner according to the present invention may solve the problems concerning a low yield and solubility and folding that conventional fusion partners have, and be widely used for protein drug and industrial protein production.

The invention relates to a DNA that enables co-expression of a protein disulfide isomerase and a polypeptide having disulfide bonds. The invention further relates to a process of producing polypeptides using the DNA and a process of enhancing the efficiency of formation of correct disulfide bond in an expression system of a gene recombinant polypeptide having disulfide bonds.

The invention discloses pichia pastoris for efficiently expressing recombinant porcine alpha1 interferon through auxiliary secretion. The pichia pastoris comprises a recombinant porcine alpha1 interferon gene segment with a DNA sequence as shown in SEQ ID No. 1 and a protein disulfide isomerase PDI gene segment with a DNA sequence as shown in SEQ ID No. 2; a multi-copy recombinant porcine alpha1 interferon gene segment and a DNA segment for inserting and expressing yeast protein disulfide isomerase are integrated in pichia pastoris; the auxiliary protein is folded in yeast cells, so that accumulation of interferon in cells is reduced, the amount of the porcine alpha 1 interferon secreted out of the cells is increased, the expression quantity of the porcine interferon alpha 1 reaches 1.43 g / L and is improved by 40% or above compared with that of a bacterial strain only integrating an interferon expression cassette, the level of producing the porcine interferon alpha 1 through thallus fermentation is remarkably improved, and the production performance of a high-copy bacterial strain is effectively exerted.

The invention provides a pronuclei fusion expression vector which comprises a nucleotide fragment. The nucleotide fragment is composed of a promoter, a gene for coding a PDI (Protein Disulfide Isomerase) parasporal protein, and a joining region which are serially connected, wherein the joining region contains an enterokinase cleavage site and a multiple cloning site. The pronuclei fusion expression vector is capable of efficiently expressing an exogenous gene in a form of a fusion protein, is good in solubility of an expression product, simple in purification, and has natural bioactivity of the exogenous gene. The pronuclei fusion expression vector is suitable for mass production of a recombinant protein, is suitable for research and production of a genetic engineering product and a biological agent, and can also be used for researching a protein function and conformation relationship.

The invention relates to the field of biotechnology and discloses a hybridoma cell, a monoclonal antibody generated by the hybridoma cell and application of the monoclonal antibody. The collection number of the hybridoma cell is CGMCC (China General Microbiological Culture Collection Center) NO.9153. The hybridoma cell and the monoclonal antibody secreted by the hybridoma cell are prepared from a member of the optimized PDI (protein disulfide isomerase) protein family as antigen protein; the hybridoma cell can stably generate the monoclonal antibody resistant to protein with an amino acid sequence as shown in SEQ ID NO:1; the monoclonal antibody has a two-way inhibition function, can inhibit thrombogenesis and blood clotting reaction, and can be applied to preparation of drugs for treating thrombotic diseases and clotting diseases.

The invention discloses a method for producing glucose oxidase. The invention firstly provides a recombinant bacterium, which is obtained by introducing a coding gene of glucose oxidase and a coding gene of a molecular chaperone into a starting bacterium. The molecular chaperone can be concretely protein disulfide isomerase and / or tuberculosis immune globulin. The invention also discloses the method for producing the glucose oxidase. The method comprises the following steps of culturing the recombinant bacterium to obtain the glucose oxidase. The invention also discloses application of the molecular chaperone in promoting microbial expression glucose oxidase. The method for producing the glucose oxidase provided by the invention has significance on secretory expression and industrial production of the enhanced glucose oxidase.

The invention belongs to the field of biomedicine and relates to recombinant engineered Escherichia coli that converts a recombinant expression vector, and the recombinant expression vector comprises genes for expressing anti-TNF antibody Fab fragment and genes for expressing protein disulfide isomerase. By expressing the ant-TNF Fab fragment with the recombinant engineered Escherichia coli, it is possible to significantly improve correct formation of disulfide bonds at the premise of significantly improving periplasmic space expression quantity.

The invention discloses an extraction method for a plant sheep embryo extract and preparation of anti-aging cosmetics. The product is prepared from sheep placenta, modified bioactive peptide, grapefruit fruit extract, paraffin, protein disulfide isomerase, a surfactant, a humectant, a preservative, propylene glycol and deionized water. Wherein the modified bioactive peptide is obtained by converting partial connected peptide bonds in the bioactive peptide into isopeptide bonds through reaction. According to the invention, the bioactive peptide in the sheep placenta is extracted and modified, so that the stability of the bioactive peptide is enhanced, the antioxidant and anti-aging effects of the bioactive peptide are enhanced, and the grapefruit fruit extract is added to enhance the auxiliary anti-aging effect. The plant sheep embryo extract and the anti-aging cosmetic disclosed by the invention can generate an anti-aging effect on the basis of cleaning the skin, so that the anti-agingeffect on the skin is achieved while the skin is cleaned.

The invention discloses an expression system of fusion protein from human serum albumin and interleukin-1 receptor antagonist. The expression system comprises a host cell and an expression vector transferred into the same. The expression vector comprises a first expression vector with an inserted fusion protein gene and a second expression vector with an inserted protein disulfide isomerase gene.The fusion protein gene comprises the human serum albumin and the interleukin-1 receptor antagonist. The host cell is Pichia pastoris GS115. The co-expression host of PDI (protein disulfide isomerase) and IGH (immunoglobulin heavy) is established, and expression index of the IGH is evidently increased. The PDI is expressed intracellularly, the IGH secretory expression is subjected to extracellular secretory expression, and accordingly no newly generated other proteins occur when concentration of the IGH in collected medium supernate increases.