77 results about "Chaperone (protein)" 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 concerns agents for the treatment of acute and chronic infections with human and animal pathogenic viruses which assemble along the cell membrane and are released through budding on the surface of the cell. Hereunto count especially causative agents of infectious diseases such as AIDS, hepatitis, hemorrhagic fever, SARS, smallpox, measles, polio or the flu. The subjects of the invention are agents that contain inhibitors of the protein folding as active components. Hereunto count inhibitors of cellular folding enzymes (the enzymatic chaperones) as well as substances that disturb the folding of proteins through chemical chaperones. The following substance classes and their derivates belong thereunto: Geldanamycin, Deoxyspergualin, 4-PBA or Herbimycin A. Due to these agents the highly organised processes of the assembly and the proteolytical maturation of virus structure proteins is disturbed. As a result the release and production of infectious decendent viruses is prevented.

A recombinant genome comprising polynucleotides encoding at least two additional molecular chaperones and a protein of interest, recombinant baculovirus vectors providing molecular chaperones and a method for producing a foreign protein using said genomes and vectors.

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 drug action mechanisms and core techniques of the present invention are summarized in figure 1. Specifically, the invention provides malignant denatured proteins, such as mutant Huntingtin proteinor alpha-synuclein, stick together to grow into oligomer aggregates (1, 2), fibrillar aggregates (3), and ultimately inclusion bodies (4). Young neuronal cells produce a large quantity of Nt-Arg through N-terminal arginylation (5) of endoplasmic reticulum chaperones, such as BiP, and thereafter, arginylated BiP (R-BiP) comes into the cytoplasm and binds with denatured proteins (6). Nt-Arg of R-BiP, as a ligand, binds with the ZZ domain of p62 (7) to induce the structural activation of p62 (8) while the ordinarily closed inactive form of p62 is changed with an open form thereof, and thus PB1 and LC3-binding domains are exposed. On the basis of oligomerization (9) by the PB1 domain, p62 binds with the denatured protein aggregates to be concentrated to autophagically degradable aggregates, that is, p62 bodies (10). Thereafter, p62 completes autophagy targeting (11) and lysosomal proteolysis through binding with LC3 protruding on the autophagosomal membranes. In young neuronal cells, theautophagic proteolysis occurring through steps 5-11 is strong, and thus the cytotoxic protein aggregates (1-5) do not accumulate, but in aged neuronal cells, the autophagic proteolysis occurring through steps 5-11 is weakened, and thus the protein aggregates (1-5) accumulate, resulting in a vicious cycle. The present invention attempts to effectively remove Huntingtin and alpha-synuclein protein aggregates and the like by artificially activating p62 using low-mass ligands of the p62 ZZ domain (12, 13). Specifically, p62 binding the ligands through step 12 promotes p62-R-BiP-denatured protein oligomerization (9) and autophagy aggregate formation (10). In addition, the ligand-62 conjugates step 13 act as autophagy activators (14), to promote LC3 synthesis, the conversion of LC3-I into LC3-II, and the like, thereby promoting the formation of autophagosomes (15).

The invention discloses novel phosphorylation sites identified in signal transduction proteins and pathways, and provides phosphorylation-site specific antibodies and heavy-isotope labeled peptides (AQUA peptides) for the selective detection and quantification of these phosphorylated sites / proteins, as well as methods of using the reagents for such purpose. Among the phosphorylation sites identified are sites occurring in the following protein types: adaptor / scaffold proteins, adhesion / extracellular matrix protein, apoptosis proteins, calcium binding proteins, cell cycle regulation proteins, chaperone proteins, chromatin, DNA binding / repair / replication proteins, cytoskeletal proteins, endoplasmic reticulum or golgi proteins, enzyme proteins, G / regulator proteins, inhibitor proteins, motor / contractile proteins, phosphatase, protease, Ser / Thr protein kinases, Protein kinase (Tyr)s, receptor / channel / cell suface proteins, RNA binding proteins, transcriptional regulators, tumor suppressor proteins, ubiquitan conjugating system proteins and proteins of unknown function.

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.

Disclosed is a method for improving folding efficiency and solubility of a target protein linked to a RNA-binding protein by using RNA molecule as a molecular chaperone, wherein the RNA molecule interacts with the RNA-binding protein. More particularly, the present invention discloses method for improving folding efficiency and solubility of a target protein by transformation of a host cell with a expression vector comprising a polynucleotide encoding the target protein linked to an RNA-binding protein; culturing the transformed host cell in an appropriate culture medium under the condition that an RNA molecule either resident inside the host cell or provided by cotransformation of the host cell with polynucleotide encoding the RNA molecule interacts with the RNA-binding protein; and purifying the soluble protein from host cell lysate. The method of the present invention is very useful for production of soluble proteins for therapeutic, prophylactic and diagnostic applications.

The invention belongs to the technical field of vaccine preparation, relates to a novel feline parvovirus vaccine, and discloses a coding gene of a feline parvovirus VP2 protein. According to the invention, an FPV VP2 protein sequence is coded and optimized through a genetic engineering technology, a recombinant plasmid is constructed, a recombinant protein is expressed by using escherichia coli, and the FPV VP2 protein is co-expressed with four molecular chaperones respectively; and a target protein is purified by different purification methods to obtain a virus-like particle which is similar to natural viruses in size and has good hemagglutination. A new thought and a new basis are provided for novel feline parvovirus vaccines.

The invention provides raccoon dog parvovirus VP2 gene, an expression vector, a recombinant bacterium, a method for preparing VP2 protein and an assembly method, and belongs to the technical field ofvaccine preparation. The raccoon dog parvovirus VP2 gene has a nucleotide sequence shown as SEQ ID No. 1. According to the invention, the raccoon dog parvovirus VP2 gene and a molecular chaperone arejointly transformed into ER2566 cells, and the molecular chaperone improves the correct folding rate of VP2 protein. The raccoon dog parvovirus VP2 gene provided by the invention can form high-qualityraccoon dog parvovirus-like particles. A raccoon dog parvovirus-like particle vaccine provided by the invention has a hemagglutination titer of 216 after being purified, and has a hemagglutinating property similar to that of a natural virus.

In one aspect, the invention provides a peptide comprising a chaperone-mediated autophagy (CMA)-targeting signal domain; a protein-binding domain that selectively binds to a target cytosolic protein; and a cell membrane penetrating domain (CMPD). In another aspect, the invention provides methods for reducing the intracellular expression level of an endogenous target protein in vitro and in an animal, wherein the method involves administration of the peptide. Methods are also provided for treating a pathological condition in an animal, the methods comprising administering the peptide to the animal. In one embodiment, the pathological condition is a neurodegenerative disease. In another embodiment of the invention, the target cytosolic protein is death associated protein kinase 1 and the CMPD is protein transduction domain of the HIV-1 Tat protein.

The invention relates to a method for purifying and preparing recombinant human insulin, in particular to a method for purifying and preparing soluble expression recombinant human insulin. The methodcomprises the following steps: firstly, designing and synthesizing a full-length sequence of recombinant human proinsulin, connecting the full-length sequence to an expression vector containing SUMO molecular chaperone peptides, introducing the constructed expression vector into a proper host cell, performing culturing, performing inducing, expressing recombinant human proinsulin fusion proteins in a soluble form, and performing purifying through affinity chromatography, enzyme digestion, ion exchange, reversed phase chromatography and other steps to finally obtain the recombinant human insulin with the purity of 98% or above. Compared with an existing method for preparing human insulin, the method has the advantages of being easy to operate, high in yield, low in production cost and the like, and is suitable for large-scale industrial production.

Disclosed is a method for improving folding efficiency and solubility of a target protein linked to a RNA-binding protein by using RNA molecule as a molecular chaperone, wherein the RNA molecule interacts with the RNA-binding protein. More particularly, the present invention discloses method for improving folding efficiency and solubility of a target protein by transformation of a host cell with a expression vector comprising a polynucleotide encoding the target protein linked to an RNA-binding protein; culturing the transformed host cell in an appropriate culture medium under the condition that an RNA molecule either resident inside the host cell or provided by cotransformation of the host cell with polynucleotide encoding the RNA molecule interacts with the RNA-binding protein; and purifying the soluble protein from host cell lysate. The method of the present invention is very useful for production of soluble proteins for therapeutic, prophylactic and diagnostic applications.