30 results about "Secretory pathway" patented technology

The invention relates to genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase and a method for constructing the same, belonging to the field of the bioengineering technology. The genetically engineered bacteria are escherichia coli BL21 (DE3) which carry two recombinant plasmids, and the recombinant plasmids are respectively plasmid pSTV28 carrying the specific genes in the Alpha-hemolysin A (hly A) pathway and plasmid pET20b(+) containing the cutinase-hly As genes. The method for constructing the genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase comprises the following steps: constructing two key recombinant plasmids and transforming the constructed recombinant plasmids in the escherichia coli BL21 (DE3) to obtain the genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase. The cutinase is produced by using the genetically engineered bacteria through culturing liquid and inducing and expressing the cutinase. The cutinase Tfu_0883 for thermophilic monospore bacteria of Thermobifida Fusca WSH03-11 is used as the report protein. The shaking flask fermentation shows that the extracellular output of the cutinase is 306U / mL which is 1.7 times of the output of the cutinase which adopts the II-type secreting pathway in the preliminary working process in the research laboratory. The cutinase is secreted and expressed efficiently.

The present invention relates to an angiogenesis inhibitor comprising meteorin as an active ingredient that is highly expressed in astrocytes of the brain and retina in the late embryonic stage and after the birth of a mouse. It is in particular highly detected in astrocyte endfeet surrounding blood vessels and promotes the expression of thrombospondin-1 / -2 (TSP-1 / -2) via autocrine pathway and thus inhibits angiogenesis. The meteorin of the present invention can be effectively used for pharmaceutical compositions and health foods that prevent vascular diseases by inhibiting angiogenesis.

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.

The invention relates to heterologous protein secreted in bacillus subtilis in a non-classical path and application thereof to protein secretory expression. Carrying no signal peptide or secretory signals, D-allulose3-epimerase RDPE from Ruminococcus sp.5-139B-FAA can be secreted out of cells in quantity; it is proved through experiments that RDPE is not secreted in an Sec or Tat path or leaked out of the cells due to cell autolysis and lysis. Therefore RDPE is non-classical secretory protein in the bacillus subtilis. RDPE is respectively fused with 18 kinds of target protein, an attempt is made to use RDPE as export signals for achieving secretory expression of the target protein, 16 kinds of fused protein are obviously expressed in the cells, 9 kinds of fused protein are successfully secreted out of the cells, and the secretion level of two kinds of fused protein accounts for more than 50% of the corresponding total protein. Therefore, a new strategy for achieving protein secretory expression in the bacillus subtilis is developed.

The present invention provides a composition for removing a hangover. In particular, the present invention discloses a method of using ajoene as an alcohol dehydrogenase inhibitor based on the fact that ajoene strongly inhibits alcohol dehydrogenase (ADH) activity. A composition for removing a hangover comprising ajoene suppresses the over-production of acetaldehyde during alcohol metabolism to maintain acetaldehyde concentration in blood at a normal level that can be degraded in the body and eliminated therefrom through normal metabolic and secretory pathways to thereby prevent the hangover after consumption of alcohol, and a method of preparing the same, are also provided. The present invention also provides a novel use of ajoene, which is a natural substance obtained by purification of garlic, and a composition for removing a hangover comprising ajoene, which is harmless to the body and strongly inhibits ADH activity, thereby showing very excellent relieving effect on the hangover.

The invention discloses a signal peptide and application thereof in production of an L-arginine recombinant bacterium by means of starch, and belongs to the fields of gene engineering and enzyme engineering. Alpha-amylase which comes from Streptomyces kathirae CCTCC M2012432 and does not carry with a self signal peptide is fused with 12 signal peptides of two main secretory pathways, namely, Sec and Tat which come from different microorganisms and then expressed in a high-yield L-arginine strain, and the signal peptide SEQ ID No.1 which can efficiently secrete the alpha-amylase is screened. It is reported for the first time that recombination corynebacterium crenatum CGMCC No.0890 / pMSPamy which is mediated to secrete the alpha-amylase can produce L-arginine through fermentation with cheap starch, and the yield of a 5-L fermentor is 48 g / L under the optimal carbon source adding condition.

A method for producing human-like glycoproteins by expressing a Class 2 α-mannosidase having a substrate specificity for Manα1,3 and Manα1,6 glycosidic linkages in a lower eukaryote is disclosed. Hydrolysis of these linkages on oligosaccharides produces substrates for further N-glycan processing in the secretory pathway.

A method for producing human-like glycoproteins by expressing a Class 2 α-mannosidase having a substrate specificity for Manα1,3 and Manα1,6 glycosidic linkages in a lower eukaryote is disclosed. Hydrolysis of these linkages on oligosaccharides produces substrates for further N-glycan processing in the secretory pathway.

This invention provides the methodology and agents for treating any disease or clinical condition which is at least partly the result of endoplasmic reticulum-associated retention of proteins. Thus, the methods and agents of the present invention provide for the release of normally retained proteins from the endoplasmic reticulum. The present invention is particularly useful for treating any disease or clinical condition which is at least partly the result of endoplasmic reticulum-associated retention or degradation of mis-assembled or mis-folded proteins.

General secretory pathway (GSP) mutant Listeria bacteria are provided. Aspects of the bacteria include the presence of a GSP mutation, e.g., a SecY and / or SecA mutation. Also provided are methods of making and using the Listeria bacteria comprising a GSP mutation as vectors and vaccines expressing a heterologous nucleic acid.

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.

The invention relates to genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase and a method for constructing the same, belonging to the field of the bioengineering technology. The genetically engineered bacteria are escherichia coli BL21 (DE3) which carry two recombinant plasmids, and the recombinant plasmids are respectively plasmid pSTV28 carrying the specific genes in the Alpha-hemolysin A (hly A) pathway and plasmid pET20b(+) containing the cutinase-hly As genes. The method for constructing the genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase comprises the following steps: constructing two key recombinant plasmids and transforming the constructed recombinant plasmids in the escherichia coli BL21 (DE3) to obtain the genetically engineered bacteria for efficiently secreting, expressing and reconstructing cutinase. The cutinase is produced by using the genetically engineered bacteria through culturing liquid and inducing and expressing the cutinase. The cutinase Tfu_0883 for thermophilic monospore bacteria of Thermobifida Fusca WSH03-11 is used as the report protein. The shaking flask fermentation shows that the extracellular output of the cutinase is 306U / mL which is 1.7 times of the output of the cutinase which adopts the II-type secreting pathway in the preliminary working process in the research laboratory. The cutinase is secreted and expressed efficiently.

The invention discloses a method for enhancing the glucose oxidase secretion by transforming folding and secreting pathway of a protein, and belongs to the technical field of gene engineering. KAR2, CNE1, ERO1, SSA4, SSO2, SEC53, BMH2, HRD1, UBC1, GCN4 and other genes in Pichia pastoris are respectively cloned and linked into pGAPZ series derived plasmids of a Pichia pastoris expression vector bya gene recombination technology, the obtained plasmids and GOD was co-expressed and transferred into Pichia pastoris GS115 strains, the effect of the strain is investigated, modular combination optimization is carried out, and the strains are screened and identified to obtain a strain having a higher glucose oxidase secretion ability than the original strains. The enzyme activity of the glucose oxidase expressed by the strain in a 3 L fermentation tank is 1972.9 U / mL. The method lays a good foundation for the large-scale production of the glucose oxidase.

The present invention relates to a eukaryotic cell containing peroxisomes that are capable to fuse with a membrane-structure of the cell involved in the secretory pathway of the cell. In this way, the eukaryotic cell is able to release the peroxisomal content outside the cell. The invention also relates to a method for production of a compound of interest in said eukaryotic cell wherein said compound of interest is present in the peroxisome of the cell. Said compound of interest will accumulate in the peroxisome by a signal promoting peroxisome localization. Preferred host cells are filamentous fungal cells.

Provided are means for evaluating and identifying putative substrates of the twin arginine translocation (Tat) secretory pathway in Streptomyces and other bacterial species. Also provided, therefore, are simple ways to express, secrete and purify correctly folded heterologous proteins on a large scale using host microorganisms, such as, Streptomyces and the Tat pathway therein. Many of the thus-produced proteins are of significant therapeutic value in the pharmaceutical and biochemical industries, particularly when they can be secreted from the host in fully-folded active form. Accordingly, there are further provided the heterologous proteins produced by the Tat secretion pathway using the foregoing methods, and the computer algorithm used to identify the Tat signal sequence and putative substrates.

This invention discloses the development of a novel platform for recombinant production of bioactive glycoproteins and cancer specific vaccines in plants. Plants and plant cell cultures have been humanized with respect to human mucin-type protein O-glycosylation. A panel of plant cell factories for production of recombinant glycoproteins with designed human O-glycosylation, including an improved cancer vaccine candidate, has been developed. The platform provides basis for i) production of an essentially unlimited array of O-glycosylated human glycoprotein therapeutics, such as human interferon α2B and podoplanin, and ii) for further engineering of additional cancer specific O-glycans on glycoproteins of therapeutical value. Currently, mammalian cells are required for human O-glycosylation, but plants offer a unique cell platform for engineering O-glycosylation since they do not perform human type O-glycosylation. Introduction of O-glycosylation into plant cells requires i) that wild-type plant cells do not modify the target peptide substrates and ii) that the appropriate enzymes and substrates are introduced into of plant cells such that O-glycosylation in the secretory pathway proceed and the glycosylated peptide substrates are preferentially exported to the exterior of the cell or accumulated in the cell. In this invention i) the integrity of transiently and stably expressed ‘mucin’ type target peptides in plants cells has been determined and ii) mucin-type O-glycosylation has been established in plants by transient and stable introduction of a Pseudomonas aeruginosa C4-epimerase, the human polypeptide GalNAc-transferases T2 and T4 (GalNAc-T2 and T4) and various human target peptides or proteins. In the present invention GalNAc-T2 and -T4 have been used to produce a Tn cancer glycoform of MUC1.

The invention provides a nucleic acid molecule encoding a growth hormone (GH) which the RSP sorting signal has been mutated, such that the GH can be constitutively secreted by the nonregulated secretory pathway (NRSP) in a mammalian cell. The invention also provides as well as a nucleic acid molecule encoding a GH in which the three-dimensional conformation of the RSP sorting signal has been altered such that the GH can be constitutively secreted by the NRSP in a mammalian cell

The invention discloses a signal peptide and application thereof in production of an L-arginine recombinant bacterium by means of starch, and belongs to the fields of gene engineering and enzyme engineering. Alpha-amylase which comes from Streptomyces kathirae CCTCC M2012432 and does not carry with a self signal peptide is fused with 12 signal peptides of two main secretory pathways, namely, Sec and Tat which come from different microorganisms and then expressed in a high-yield L-arginine strain, and the signal peptide SEQ ID No.1 which can efficiently secrete the alpha-amylase is screened. It is reported for the first time that recombination corynebacterium crenatum CGMCC No.0890 / pMSPamy which is mediated to secrete the alpha-amylase can produce L-arginine through fermentation with cheap starch, and the yield of a 5-L fermentor is 48 g / L under the optimal carbon source adding condition.