Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Patent
1994-09-02
1998-01-13
Hendricks, Keith D.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
530333, C07K 100
Patent
active
057081409
DESCRIPTION:
BRIEF SUMMARY
The invention lies in the field of genetic engineering and, in particular, is concerned with the use of 7B2 as chaperone in vivo or in vitro. The invention accordingly concerns a method for producing a desired protein in vivo with the aid of recombinant cells capable of expressing 7B2 and of expressing and secreting said desired protein. Another aspect is accordingly an in vitro method for the deaggregation or prevention of aggregation of protein by treating the protein with 7B2.
BACKGROUND OF THE INVENTION
Different transport mechanisms for secretory proteins exist within the eukaryotic cell. All secretory cells arc capable of releasing secretory proteins by protein synthesis at the endoplasmatic reticulum (ER), transport via ER to the Golgi apparatus and from the Golgi apparatus to so-called transport granules. The membrane of a transport granule fuses with the plasma membrane after which the secretory proteins can leave the cell by exocytosis. When secretory proteins are made they are also released, in other words there is no regulation of protein release. This protein transport is subsequently designated the constitutive secretory route and is present in all eukaryotic cells including yeast. Prokaryotic cells also have a not regulated, i.e. constitutive, route of protein secretion.
The release of certain secretory proteins is known to be regulated in a restricted group of higher eukaryotic cells. These cells comprise neurons and endocrine cells, in other words cells producing precursors of bioactive proteins. The synthesis in these cells takes place at the ER, transport goes from ER to Golgi as in the constitutive cells but then proceeds from Golgi to secretory granules. In these secretory granules the correct environment is present for the specific post-translational modifications of the precursors. This type of protein transport is indicated as the regulated route. The exocytosis of the bioactive proteins only occurs when the cell undergoes a correct external stimulus. This type of cell is indicated as a regulated secretory cell.
Correct folding of proteins produced by a cell is ensured by molecular chaperone proteins which are essential for correct functioning of cells as they ensure that proteins produced by the cells are folded in a correct manner. Correct folding is necessary for correct transport of these proteins to the right destination within the cell. Thus, chaperones are not only necessary for correct protein folding but also for correct protein transport. Specific chaperone proteins have e.g. been described for protein transport from the cell cytoplasm to cell organelles such as the mitochondrion, the chloroplast, the cell nucleus and the ER. The production of many of these proteins is induced in cells that are subjected to a heat shock and chaperone proteins are therefore often called "heat-shock proteins".
The production of recombinant bioactive protein has become possible through developments in the field of recombinant DNA technology, but is often not successful in practice because frequently folding and/or secretion of the heterologous proteins that are to be produced does not occur correctly or occurs inefficiently. Such a problem usually occurs in cases of strong expression of the heterologous protein in a cell which has insufficient capacity to ensure correct folding and optionally secretion of a protein that is produced in a large amount. The problem arises from the fact that recombinant proteins are produced in large amounts in prokaryotic cells or eukaryotic cells that so that the activity of the cellular folding proteins is not sufficient for folding the huge amount of foreign protein. Additional problems arise if recombinant proteins are desired to be processed and secreted but the host cells comprise only to an insufficient extent a route of protein transport. Moreover, if proproteins such as prohormones or precursors of neuropeptides are intended to be produced in higher eukaryotic cells which proproteins are desired to be processed in the cell to bioactive proteins, prob
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Chaudhuri Bhabatosh
Martens Gerardus Julianus Maria
Stephan Christine
Ciba-Geigy Corporation
Hendricks Keith D.
Nowak Henry P.
Spruill W. Murray
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