Method for the manufacture of recombinant trypsin
AssigneePolymun Scientific Immunbiologische Forschung GmbH
InventorsDiethard Mattanovich, Hermann Katinger, Hubertus Hohenblum, Stefan Naschberger, Robert Weik
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Abstract
The present invention relates to a method for the manufacture and purification of recombinant trypsinogen and trypsin in E. coli and yeast, using high yield expression vectors with and without secretion leader sequences. The invention further relates to an improved method and apparatus for carrying out protein refolding specifically useful for processing trypsinogen that has accumulated intracellularly in the form of inclusion bodies.
Claims16
A method for the manufacture of recombinant trypsinogen or trypsin comprising incubating transformed host cells expressing a trypsinogen gene in a suitable cell culture nutrient medium at conditions allowing for cell multiplication and simultaneous or subsequent expression of the trypsinogen gene, separating intracellularly or extracellularly accumulated trypsinogen from the cell suspension, and optionally further processing the separated trypsinogen, characterized in that the method comprises incubating the transformed host cells at an acidic pH at or above pH 2, the transformed host cells being yeast cells transformed with an expression vector that contains a naturally occurring trypsinogen gene, and after incubation, during which no trypsinogen activation or trypsin activity is detected in the medium by SDS polyacrylamide gel electrophoresis, separating the cells from the medium and obtaining a solid-free supernatant containing correctly folded trypsinogen.
Dependencies are inferred from the claim text.