Roche Diagnostics Operations, Inc.

Roche Diagnostics Gmbh

F. Hoffmann-La Roche Ag

Werner Hoelke

Johann-Peter Thalhofer

Rainer Mueller

Frank Geipel

Thomas Kirschbaum

Markus Weber

Antje Liehre

Bernhard Suppmann

Stephan Glaser

Hellmut Eckstein

Michaela Fischer

Helmut Lenz

Harald Sobek

Helmut Burtscher

Manfred Schmidt

Jose Luis Millan

Bettina Bommarius

Rainer Muller

Helmut Schoen

Wolfgang Hoesel

Kirsten Sonn

Beata Kanne

Thomas Marx

Artur Hoffmann

Described are compositions, in particular lyophilizates, containing proteolytic enzymes, and methods for producing the compositions. Typically these compositions contain one or more proteases with collagenase activity and a neutral protease, for example, thermolysin. The compositions are free of acetate salts. Surprisingly, such compositions can be dissolved in water more rapidly than lyophilized protease mixtures of the state of the art.

Methods of preparing a solid composition containing proteases

The present invention deals with the proteolytic enzyme thermolysin which tends to be unstable in aqueous solution. The invention provides methods and compositions to enhance the stability of dissolved thermolysin in aqueous solution. Thermolysin, crude thermolysin or a lyophilisate containing thermolysin and one or more salts, is contacted with an aqueous buffer with a low salt concentration and a first solution is formed. Subsequently, a further salt in solid form is added and dissociated, thereby forming a second solution comprising thermolysin in a stabilized form.

Stabilization of thermolysin in aqueous solution

The present invention deals with the proteolytic enzyme thermolysin which tends to be unstable in aqueous solution. The invention provides methods and compositions to enhance the stability of dissolved thermolysin in aqueous solution. Thermolysin, crude thermolysin or a lyophilizate containing thermolysin and one or more salts, is contacted with an aqueous buffer with a low salt concentration and a first solution is formed. Subsequently, a further salt in solid form is added and dissociated, thereby forming a second solution comprising thermolysin in a stabilized form.

The present invention provides an aqueous composition comprising a protein with enzymatic activity of alpha-galactosidase. The present invention further provides a method of stabilizing an aqueous composition comprising a protein with enzymatic activity of alpha-galactosidase, and a method of preparing a purified aqueous composition comprising the protein with enzymatic activity of alpha-galactosidase.

Stabilized aqueous alpha-galactosidase composition and methods relating thereto

The present invention provides improved media for the cultivation ofand culture supernatants for the biotechnological production of collagenase enzymes. The nutrient media according to the invention comprise one or more peptones from a non-mammalian source, preferably plant-derived peptones. The media can additionally comprise fish gelatin. The invention provides media, culture supernatants comprisingcollagenase, and methods to produce said collagenase.

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The present invention provides a method for purifyingcollagenase type I and type II proteins from a complex mixture by subsequently performing a precipitation with ammonium sulfate, hydrophobic interaction chromatography, cation exchange chromatography, and anion chromatography. Conditions are provided which lead to a stabilized, partially purified preparation even after the precipitation step. The method of the invention leads to a quick and efficient removal of other proteolytic activities. The preparations according to the invention provide exceptionally pure and intact collagenase type I and type II proteins which are enzymatically active. The invention also provides blends of the two isolated proteins. The invention further provides the use of the purified collagenase proteins or blends thereof for treating a tissue sample in vitro.

By deglycosylating an enzyme derived from a eukaryotic organism prior to forming a conjugate, i.e. prior to attaching the enzyme to a molecule capable of binding to a target molecule, the sensitivity of an assay for detecting the presence or determining the quantity of a target molecule, in which the conjugate is used as a labelled component, can be increased. The present invention provides conjugates, methods for providing the same, use of such conjugates as well as kits containing the conjugates.

Deglycosylated enzymes for conjugates

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.

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

The invention concerns a DNA coding a eukaryotic highly active alkaline phosphatase with a specific activity of more than 3000 U/mg. The invention also concerns a process for the production of a DNA according to the invention, a vector containing the DNA according to the invention and a cell line containing this vector. Furthermore the invention concerns a recombinant highly active alkaline phosphatase with a specific activity of more than 3000 U/mg which is coded by the DNA according to the invention.

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Penzberg, Germany

Werner Hoelke