N.v. Organon

Msd Oss B.v.

Peter Van Der Meijden

Lubbert Dijkhuizen

Robert Van Der Geize

Gerda Hessels

Gijsbert Willem Karel Van Dedem

Michel Hendrikus Maria Eppink

Roeland Wilhelmus Wassenaar

Michel Hendrikus Eppink

Gijsbert Willem Van Dedem

The present invention relates to a method for the isolation of proteins that comprise disulfide-bonds in their native conformation. Essentially, a method of the present makes the use of reducing agents such as &#x3b2;-mercaptoethanol or dithiothreitol in protein isolation methods obsolete. A method of the present invention is particularly suitable for the isolation of precursor proteins such as proinsulin from recombinant cells.

Method for protein isolation in anoxic conditions

The present invention relates to a method for the isolation of proteins that comprise disulfide bonds in their native conformation. Essentially, a method of the present invention makes use of reducing agents such as &#x3b2;-mercaptoethanol or dithiothreitol in protein isolation methods obsolete. A method of the present invention is particularly suitable for the isolation of precursor proteins such as proinsulin from recombinant cells.

The invention relates to an isolated polynucleotide sequence comprising a nucleic acid sequence encoding the amino acid sequence of KshA protein or of KshB protein, encoded by nucleotides 499-1695 of SEQ ID NO:1 or by nucleotides 387-1427 of SEQ ID NO:2, respectively, and functional homologues thereof. The polynucleotides of the invention can be used to construct genetically modified microorganisms blocked in 3-ketosteroid 9&#x3b1;-hydroxylase activity, which are useful in the microbial degradation of steroids to accumulate certain steroid products.

Identification of 3-ketosteroid 9-alfa-hydroxylase genes and microorganisms blocked in 3-ketosteroid 9-alfa-hydroxylase activity

Indentification of 3-ketosteroid 9-alfa-hydroxylase genes and microorganisms blocked in 3-ketosteroid 9-alfa-hydroxylase activity

The invention relates to an isolated polynucleotide sequence comprising a nucleic acid sequence encoding the amino acid sequence of KshA protein or of KshB protein, encoded by nucleotides 499&#x2013;1695 of SEQ ID NO: 1 or by nucleotides 387&#x2013;1427 of SEQ ID NO:2, respectively, and functional homologues thereof. The polynucleotides of the invention can be used to construct genetically modified microorganisms blocked in 3-ketosteroid 9&#x3b1;-hydroxylase activity, which are useful in the microbial degradation of steroids to accumulate certain steroid products.

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Oss, Netherlands

Peter Van Der Meijden