21-hydroxylation of steroids
AssigneeSanofi
InventorsClaus Lattemann, Thomas Stillger, Bernd Janocha, Hans-Falk Rasser, Sebastian Rissom, Simone Anderko, Rita Bernhardt, Frank Hannemann
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Abstract
Generally, the present invention relates to the field of steroid hydroxylation. More specifically, the present invention relates to a method for the 21-hydroxylation of steroids in cells. It also relates to cells expressing a steroid 21-hydroxylating enzyme or steroid 21-hydroxylase, expression vectors comprising a nucleic acid encoding for a steroid 21-hydroxylase and a kit for carrying out the method for the 21-hydroxylation of steroids in cells.
Claims20
A process for the hydroxylation of the carbon atom 21 of a steroid, comprising the steps of:
- (a) providing a cell expressing:
- (i) a heterologous CYP21A2 protein or a functional variant thereof;
- (ii) at least one heterologous electron transfer system capable of transferring electrons to CYP21A2, wherein the at least one electron transfer system is selected from the group consisting of:
- the combination of a flavodoxin reductase (Fpr) and an adrenodoxin;
- the combination of a Fpr and the ferredoxin domain of an electron transfer domain (etpfd);
- the combination of an adrenodoxin reductase homolog (Arh) and an adrenodoxin;
- and
- the combination of an Arh and etpfd;
- and
- (iii) one or more chaperones facilitating folding of CYP21A2;
- and
- (b) adding the steroid to the cell.
A cell expressing:
- (i) a heterologous CYP21A2 protein or a functional variant thereof;
- (ii) at least one heterologous electron transfer system capable of transferring electrons to CYP21A2, wherein the at least one electron transfer system is selected from the group consisting of:
- the combination of a flavodoxin reductase (Fpr) and an adrenotoxin;
- the combination of a Fpr and the ferredoxin domain of an electron transfer domain (etpfd);
- the combination of an adrenotoxin reductase homolog (Arh) and an adrenotoxin;
- and
- the combination of an Arh and etpfd;
- and
- (iii) one or more chaperones facilitating folding of CYP21A2.
A multicistronic expression vector comprising:
- (i) a nucleic acid encoding for a CYP21A2 protein or a functional variant thereof;
- (ii) one or more nucleic acids encoding for at least one heterologous electron transfer system capable of transferring electrons to CYP21A2, wherein the at least one electron transfer system is selected from the group consisting of:
- the combination of a flavodoxin reductase (Fpr) and an adrenotoxin;
- the combination of a Fpr and the ferredoxin domain of an electron transfer domain (etpfd);
- the combination of an adrenotoxin reductase homolog (Arh) and an adrenotoxin;
- and
- the combination of an Arh and etpfd;
- and optionally
- (iii) one or more nucleic acids encoding for chaperones facilitating folding of CYP21A2.
Dependencies are inferred from the claim text.