Roche Diagnostics Operations, Inc.

Sanofi-a Ventis Deutschland Gmbh

Roche Diagnostics Gmbh

F. Hoffmann-la Roche Ag

Frank Geipel

Johann-Peter Thalhofer

Rainer Mueller

Stephan Glaser

Werner Hoelke

Thomas Kirschbaum

Hellmut Eckstein

Claus S Schneider

Stephanie Ronning

Bernhard Rexer

Rainer Muller

Michael Ratka

Claudia Giessel

Erhard Kopetzki

Frank Zocher

Bettina Bommarius

Helmut Schoen

Christoph Hoh

Claus Schneider

Elvira Wolf

The present invention relates to the production of a variant of recombinant trypsin with increased substrate specificity for arginine versus lysine in non-animal host organisms. Moreover, the present invention relates to a variant of recombinant trypsin and their production. Also provided are use of recombinant porcine pancreatic trypsin variants for cleavage of precursors of insulins, and kits containing the variant of trypsin.

Cleavage of precursors of insulins by a variant of trypsin

The present invention concerns a method for producing recombinant trypsin from porcine pancreas inwhich is soluble and secreted into the culture medium, whereby expression at pH 3.0-4.0 substantially prevents activation of trypsinogen to &#x3b2;-trypsin and autolysis of &#x3b2;-trypsin by &#x3b1;-trypsin into &#x3b5;-trypsin and from there into inactive peptides.

Method for producing recombinant trypsin

The invention concerns a method for the expression of a gene coding for a soluble proteinase K in yeast e.g. inwith subsequent secretion into the culture medium. In addition a method for purifying the heterologously expressed and secreted proteinase K is described.

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The invention concerns a method for the recombinant production or expression of eukaryotic alkaline phosphatase mutants in yeast cells wherein the specifically introduced mutations result in a reduction of the specific AP activity by at least a factor 1:100. The invention also concerns a method for inserting corresponding nucleic acid sequences into a vector for expression in methylotrophic yeast strains and it concerns corresponding vectors and host strains.

Production of weakly active or inactive mutants of alkaline phosphatase and their expression in yeast

This invention involves a process for selecting and producing eukaryotic alkaline phosphatase in yeast. Yeast cells are subjected to multiple transformations using a vector comprising a first resistance marker gene and the alkaline phosphatase gene. Those strains that grow in media containing the first resistance marker are further transformed using a vector comprising a second selection marker gene and the alkaline phosphatase gene. Transformants that grow in media containing the second selection marker are selected for expressing the eukaryotic alkaline phosphatase.

Expression of alkaline phosphatase in yeast

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Penzberg, Germany

Frank Geipel