Roche Diagnostics Operations, Inc.

Roche Diagnostics Gmbh

F. Hoffmann-la Roche Ag

Johann-Peter Thalhofer

Werner Hoelke

Rainer Mueller

Frank Geipel

Harald Sobek

Bernhard Suppmann

Manfred Schmidt

Stephan Glaser

Thomas Kirschbaum

Hellmut Eckstein

Markus Weber

Antje Liehre

Michael Greif

Helmut Schoen

Bernhard Rexer

Stephanie Ronning

Claus S Schneider

Rainer Muller

Claudia Giessel

Michael Ratka

Michaela Fischer

Christian Rudolph

Rainer Schmuck

Bruno Frey

Bettina Bommarius

Ulrike Fischer

Armin Ruf

Markus Pajatsch

Kirsten Sonn

Guenter Guertler

Peter Pallua

Rainer Müller

Artur Hoffmann

Thomas Marx

Elvira Wolf

Claus Schneider

Hoelke Werner

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 relates to a formulation of a thermostable DNA polymerase which is completely free of detergents and its particular use in real time polymerase chain reaction (PCR). Such a formulation may be obtained if the selected purification method does not require the addition of a detergent at any purification step.

Detergent free polymerases

The present disclosure provide novel variants of T7 RNA polymerase. Embodiments of T7 variants, according to the instant invention, include a Cysteine-Serine substitution on position 723 of the amino acid sequence of the T7 polypeptide. Embodiments of T7 variants according to the instant invention have a DNA-dependent RNA polymerase enzymatic activity and a reduced tendency to form intramolecular homodimers by way of oxidizing thiol groups. The amino acid substitutions within the T7 variants disclosed herein impact minimally, if at all, the RNA polymerase activity of the T7 polypeptide. Further, the mutations of the disclosed embodiments may optionally be combined with mutations which provide enhanced thermostability compared to the wild-type reference.

T7 RNA polymerase variants with cysteine-serine substitutions

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 provides improved variants of T7 RNA polymerase by introducing novel mutations which lead to improved thermostability of the enzyme. According to the invention, amino acid substitutions at the positions Val426, Ser633, Val650, Thr654, Ala702, Val795, and combinations thereof are advantageous.

T7 RNA polymerase variants with enhanced thermostability

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.

A method for producing solid proteinase K which is insoluble in water and has high purity, the method comprising the steps of adding to an aqueous solution of proteinase K ammonium sulfate in an amount of 0.1 to 0.2 M per 5 minutes up to a final amount of 1.5 to 2 M after 1 hour, thereby precipitating the proteinase K as a solid, and isolating the solid proteinase K.

Highly purified proteinase K

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.

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.

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Weilheim, Germany

Johann-Peter Thalhofer