Expression system for recombinant proteins
AssigneeGenzyme Corporation
InventorsNick Wan, Henry Hoppe IV, Jason C. Goodrick, Bernhard M. Schilling
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Abstract
A continuous fermentation process has been developed in Pichia pastoris (P. pastoris) in order to produce large quantities of recombinant human proteins. High expression levels have been demonstrated in continuous production of the enzyme by P. pastoris with a constitutive promoter in a 1.5-liter working volume fermenter using either glucose or glycerol as the carbon source. The fermentation could be extended for long periods of time with a excellent steady-state protein concentration and cell densities achieved. No proteolytic degradation of the enzyme was seen in the continuous fermentation mode.
Claims17
A method for the production of recombinant proteins with high-mannose carbohydrate structure, comprising continuously culturing cells of Pichia pastoris, which cells comprise a DNA molecule which encodes a protein of interest, under conditions suitable for the expression of said DNA molecule.
A method for the production of recombinant glucocerebrosidase with high-mannose carbohydrate structure, comprising culturing cells of Pichia pastoris which cells comprise a DNA molecule which encodes glucocerebrosidase, under conditions suitable for the expression of said DNA molecule.
A method for purification of recombinant human glucocerebrosidase with high-mannose carbohydrate structure, comprising culturing cells of Pichia pastoris which cells comprise a DNA molecule which encodes glucocerebrosidase, under conditions suitable for the expression of said DNA molecule to produce recombinant human glucocerebrosidase in a cell culture, and purifying said produce recombinant human glucocerebrosidase from said cell culture.
A method for the production of recombinant sphingomyelinase with high-mannose carbohydrate structure, comprising culturing cells of Pichia pastoris which cells comprise a DNA molecule which encodes sphingomyelinase, under conditions suitable for the expression of said DNA molecule.
A method for purification of recombinant human sphingomyelinase with high-mannose carbohydrate structure, comprising culturing cells of Pichia pastoris which cells comprise a DNA molecule which encodes sphingomyelinase, under conditions suitable for the expression of said DNA molecule to produce recombinant human sphingomyelinase in a cell culture, and purifying said produce recombinant human sphingomyelinase from the cell culture.
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