Method
OwnerClaus Lindvald JOHANSEN, Soren Kjaerulff +4inventors
InventorsClaus Lindvald JOHANSEN, Soren Kjaerulff, Susan Mampusta Madrid, Henrik Pedersen, Charlotte Horsmans Poulsen, Masoud Rajabi Zargahi
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Abstract
A method is described for releasing a soluble or membrane associated intracellular protein of interest (POI) comprising the steps of: providing a cell comprising a soluble or membrane associated intracellular POI; contacting the cell with a membrane extracting composition; and causing the POI to be released from the cell under conditions sufficient for the specific release of the POI and in a soluble form.
Claims43
A method for extracting a soluble or membrane associated intracellular recombinant protein of interest (POI) from a bacterial, yeast or fungal cell comprising the steps of:
- (a) providing a bacterial, yeast or fungal cell comprising a soluble or membrane associated intracellular recombinant POI;
- (b) releasing the recombinant POI from the cell by contacting the cell with a membrane extracting composition comprising a quarternary ammonium compound at a concentration of between 0.05% to 0.6% by weight, under conditions sufficient for the release of the recombinant POI and in a soluble form
- (c) recovering the recombinant POI from the membrane extracting composition.
A method of using a membrane extracting composition comprising a quaternary ammonium compound to extract a soluble or membrane associated intracellular recombinant POI from a bacterial, yeast or fungal cell, in which the membrane extracting composition comprises a quaternary ammonium compound at a concentration of between 0.05% to 0.6% by weight and is contacted with the bacterial, yeast or fungal cell, and in which the released recombinant POI is recovered from the membrane extracting composition.
A method for screening for mutated cells or transformed cells producing elevated levels of a soluble or membrane associated intracellular recombinant POI comprising the steps of:
- (a) growing the mutated cells at 30° C.;
- (b) incubating the mutated cells or transformed cells with the membrane extracting composition comprising a quaternary ammonium compound at a concentration of between 0.05% to 0.6% by weight;
- (c) recovering the cell free medium
- (d) screening the cell free medium for elevated levels of the intracellular recombinant POI;
- such that the presence of the intracellular recombinant POI in the cell free medium is indicative that the intracellular recombinant POI has been released.
A membrane extracting composition suitable for releasing a soluble or membrane associated intracellular recombinant POI, wherein the composition is contacted with the cell under the following conditions:
- (a) a percentage by weight of quaternary ammonium compound from about 0.05% to about 0.6%;
- (b) a pH of from about 2.0 to about 11.0
- (c) a temperature of from about 4° C. to about 40° C.;
- Such that the intracellular recombinant POI substantially free of contaminating proteins is obtained.
A method of using a membrane extracting composition comprising a quaternary ammonium compound to extract a soluble or membrane associated intracellular recombinant POI from a bacterial, yeast or fungal cell, the POI being released from the bacterial, yeast or fungal cell, in which the membrane extracting composition comprises a quaternary ammonium compound at a concentration of between 0.05% to 0.6% by weight and is contacted with the bacterial, yeast or fungal cell and in which the released recombinant POI is recovered from the membrane extracting composition.
A substantially non-glycosylated POI released from a eukaryotic host organism.
Dependencies are inferred from the claim text.