- 专利标题： SYNTHESIS OF FUNCTIONAL CYTOCHROME P450 MONOOXYGENASES USING A EUKARYOTIC CELL-FREE SYSTEM COMPRISING MEMBRANE VESICLES
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申请号： EP22192384.0申请日： 2022-08-26
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公开(公告)号： EP4328302A1公开(公告)日： 2024-02-28
- 发明人: KNAUER, Jan, Felix , KUBICK, Stefan , ZEMELLA, Anne
- 申请人： Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
- 申请人地址： DE 80686 München Hansastraße 27c
- 代理机构： v. Bezold & Partner Patentanwälte - PartG mbB
- 主分类号： C12N9/02
- IPC分类号： C12N9/02 ; C12P21/02
摘要：
The invention relates to a method for preparing functional membrane attached monooxygenases in cell-free systems which comprises at least the following steps:
a) providing a reaction medium comprising at least the following components:
a nucleic acid template coding for a Cytochrome P450 monooxygenase classified as EC 1.14.-.-, an RNApolymerase, a eukaryotic cell lysate containing ER-derived membrane vesicles which comprise NADPH-Cytochrom-P450-oxidoreductase (CPR), optionally heme or heme derivatives, in particular heme complexes selected from the group comprising or consisting of hematin and hemin;
b) performing a cell-free reaction to synthesize the monooxygenase protein, wherein the cell-free translation reaction is performed at a temperature in the range from 18 to 33°C.
The invention further relates to a reaction medium for preparing a functional Cytochrome P450 monooxygenase classified as EC 1.14.-.- in a cell-free system comprising at least the following components:
a nucleic acid template coding for a Cytochrome P450 classified as EC 1.14.-.-, an RNA polymerase, a eukaryotic cell lysate containing membrane vesicles which comprise NADPH-Cytochrom-P450-oxidoreductase (CPR), heme or heme derivatives, in particular heme complexes selected from the group comprising or consisting of hematin and hemin.
a) providing a reaction medium comprising at least the following components:
a nucleic acid template coding for a Cytochrome P450 monooxygenase classified as EC 1.14.-.-, an RNApolymerase, a eukaryotic cell lysate containing ER-derived membrane vesicles which comprise NADPH-Cytochrom-P450-oxidoreductase (CPR), optionally heme or heme derivatives, in particular heme complexes selected from the group comprising or consisting of hematin and hemin;
b) performing a cell-free reaction to synthesize the monooxygenase protein, wherein the cell-free translation reaction is performed at a temperature in the range from 18 to 33°C.
The invention further relates to a reaction medium for preparing a functional Cytochrome P450 monooxygenase classified as EC 1.14.-.- in a cell-free system comprising at least the following components:
a nucleic acid template coding for a Cytochrome P450 classified as EC 1.14.-.-, an RNA polymerase, a eukaryotic cell lysate containing membrane vesicles which comprise NADPH-Cytochrom-P450-oxidoreductase (CPR), heme or heme derivatives, in particular heme complexes selected from the group comprising or consisting of hematin and hemin.
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