Novo Nordisk A/s

Novozymes A/s

Novo Industri A/s

Fanny Norris

Kjeld Norris

Lars Christian Petersen

So Slashedren Bjo Slashedrn

Dorrit A Aaslyng

Sven Branner

Sven Hastrup

Villy Johannes Jensen

Ole Hvilsted Olsen

Claus Bregengaard

Leif No slashedrskov-Lauritsen

Niels D Christensen

Viggo Diness

Leif N&oslash;rskov-Lauridsen

Soren E Bjo Slashedrn

Lars Thim

Steffen Bj&oslash;rn Petersen

Steffen Bjorn Petersen

Donald C Foster

Cindy A Sprecher

Per Franklin Nielsen

Soren Erik Bjorn

Mogens Christensen

Alister J Moody

Mogens T Hansen

Leif Norskov-Lauridsen

Leif No slashedrskov-Lauridsen

Steffen Bjo Petersen

The present invention relates to mutations of a subtilisin gene which result in changes in the chemical characteristics of subtilisin enzymes. Mutations at specific nucleic acids of the subtilisin gene result in amino acid substitutions and consequently, altered enzyme function. Some of these mutant enzymes exhibit physical properties advantageous to industrial applications, particularly in the detergent industry, providing subtilisin which is more stable to oxidation, possesses greater protease activity, and exhibits improved washability.

Useful mutations of bacterial alkaline protease

The present invention relates to mutations of the subtilisin gene, some of which result in changes in the chemical characteristics of subtilisin enzyme. Mutations are created at specific nucleic acids of the subtilisin gene and, in various specific embodiments, the mutant enzymes possess altered chemical properties including, but not limited to, increased stability to oxidation, augmented proteolytic activity, and improved washability. The present invention also relates, but is not limited to, the amino acid and DNA sequences for two proteases derived from variants, subtilisin 147 and subtilisin 309, as well as mutations of these genes and the corresponding mutant enzymes.

Human spasmolytic polypeptide in glycosylated form

The present invention relates to mutations of the subtilisin gene, some of which result in changes in the chemical characteristics of subtilisin enzyme. Mutations are created at specific nucleic acids of the subtilisin gene and, in various specific embodiments, the mutant enzymes possess altered chemical properties including, but not limited to, increased stability to oxidation, augmented proteolytic activity, and improved washability. The present invention also relates, but is not limited to, the amino acid and DNA sequences for two proteases derived from Bacillus lentus variants, subtilisin 147 and subtilisin 309, as well as mutations of these genes and the corresponding mutant enzymes.

A variant of the C-terminal Kunitz-type protease inhibitor domain of the .alpha.3 chain of human type VI collagen, the variant comprising the following amino acid sequence X.sup.1 X.sup.16 Asp Ile Cys Lys Leu Pro Lys Asp X.sup.2 Gly X.sup.3 Cys X.sup.4 X.sup.5 X.sup.6 X.sup.7 X.sup.8 X.sup.9 Trp Tyr Tyr Asp Pro Asn Thr Lys Ser Cys Ala Arg Phe X.sup.10 Tyr Gly Gly Cys X.sup.11 X.sup.12 X.sup.13 Glu Asn Lys Phe X.sup.14 Ser Gln Lys Glu Cys Glu Lys Val Cys Ala Pro X.sup.15 (SEQ ID NO. 1) wherein X.sup.1, X.sup.15, and X.sup.16 represents a naturally occurring amino acid residues except Cys and X.sup.2 -X.sup.14 each independently respresents a naturally occurring amino acid residue, with the proviso that at least one of the amino acid residues X.sup.1 -X.sup.16 is different from the corresponding amino acid residue of the native sequence. Alternatively, the N-terminal Asp may be preceded by H or 3-5 amino acid residues and the C-terminal Pro may be followed by OH or 3-5 amino acid residues.

Human Kunitz-type protease inhibitor variants

The present invention relates to methods for producing aprotinin and analogs thereof in yeast, synthetic genes encoding such products, expression vectors and transformed yeast cells. The invention further relates to aprotinin analogs, particularly analogs with increased specific inhibitory activity and/or reduced nephrotoxicity compared to native aprotinin, as well as compositions comprising such analogs.

Aprotinin analogs

The present invention relates to a human Kunitz-type protease inhibitor comprising the following amino acid sequence

Human kunitz-type protease inhibitor and variants thereof

Process for preparing aprotinin and aprotinin analogs in yeast cells

Variant of human Kunitz-type protease inhibitor domain II of tissue factor pathway inhibitor (TFPI), the variant comprising the following amino acid sequence ##STR1## wherein Xaa at position 1 is H or a naturally occurring amino acid residue except Cys, each Xaa at positions 2-5 is independently a naturally occurring amino acid residue except Cys or is absent, each Xaa at positions 14, 16, 18, 19, 20, 21, 22, 23, 37, 42, 43, 44, and 49 independently a naturally occurring amino acid except Cys, each Xaa at positions 61, 62, 63, and 64 is independently a naturally occurring amino acid except Cys or is absent, and Xaa at position 65 is OH or a naturally occurring amino acid except Cys, with the proviso that at least one of the amino acid residues designated Xaa is different from the amino acid residue of the native sequence.

Human Kunitz-type protease inhibitor variant

A polypeptide for production in yeast comprises a fusion of a signal peptide, a leader peptide and a heterologous protein or polypeptide. The polypeptide is modified in its amino acid sequence adjacent to a yeast processing site positioned between the C-terminal end of the leader peptide and the N-terminal end of the heterologous protein so as to provide a presentation of the processing site which makes it accessible to proteolytic cleavage. Such a presentation is provided by adding one or more amino acids (at least one of which is negatively charged) to either the C-terminal end of the leader or the N-terminal end of the protein, or both. The heterologous protein may, for instance, be aprotinin or insulin precursor or an analogue thereof.

Yeast processing system

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Hellerup, Denmark

Fanny Norris