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    Aspergillus ochraceus 11 alpha hydroxylase and oxidoreductase
    1.
    发明申请
    Aspergillus ochraceus 11 alpha hydroxylase and oxidoreductase 失效
    曲霉曲霉11α羟化酶和氧化还原酶

    公开(公告)号：US20050003473A1

    公开(公告)日：2005-01-06

    申请号：US10900856

    申请日：2004-07-28

    申请人： Suzanne Bolten ,  Robert Clayton ,  Alan Easton ,  Leslie Engel ,  Dean Messing ,  John Ng ,  Beverly Reitz ,  Mark Walker ,  Ping Wang

    发明人： Suzanne Bolten ,  Robert Clayton ,  Alan Easton ,  Leslie Engel ,  Dean Messing ,  John Ng ,  Beverly Reitz ,  Mark Walker ,  Ping Wang

    IPC分类号： G01N33/536 ,  A61K35/74 ,  A61P9/00 ,  A61P19/02 ,  A61P29/00 ,  A61P35/00 ,  C07K1/02 ,  C07K14/38 ,  C07K16/40 ,  C07K19/00 ,  C12N1/14 ,  C12N1/15 ,  C12N1/19 ,  C12N1/21 ,  C12N3/00 ,  C12N5/10 ,  C12N9/02 ,  C12N15/09 ,  C12P21/08 ,  C12P33/10 ,  C12Q1/26 ,  G01N33/573 ,  C12P33/06 ,  C07H21/04 ,  C12N1/16 ,  C12N15/74 ,  C12P33/08

    CPC分类号： C12N9/0083 ,  C07K14/38 ,  C07K16/40 ,  C12N1/14 ,  C12N3/00 ,  C12N9/0004 ,  C12N2799/026 ,  C12P33/10

    摘要： The present invention relates to a novel cytochrome P450-like enzyme (Aspergillus ochraceus 11 alpha hydroxylase) and an oxidoreductase (Aspergillus ochraceus oxidoreductase) isolated from cDNA library generated from the mRNA of Aspergillus ochraceus spores. When the cDNA encoding the 11 alpha hydroxylase was co-expressed in Spodoptera frugiperda (Sf-9) insect cells with the cDNA encoding human oxidoreductase as an electron donor, it successfully catalyzed the conversion of the steroid substrate 4-androstene-3,17-dione (AD) to 11 alpha-hydroxy-AD as determined by HPLC analysis. The invention also relates to nucleic acid molecules associated with or derived from these cDNAs including complements, homologues and fragments thereof, and methods of using these nucleic acid molecules, to generate, for example, polypeptides and fragments thereof. The invention also relates to the generation of antibodies that recognizes the A. ochraceus 11 alpha hydroxylase and oxidoreductase and methods of using these antibodies to detect the presence of these native and recombinant polypeptides within unmodified and transformed host cells, respectively. The invention also provides methods of expressing the Aspergillus 11 alpha hydroxylase gene separately, or in combination with human or Aspergillus oxidoreductase, in heterologous host cells, to facilitate the bioconversion of steroid substrates to their 11 alpha hydroxy-counterparts.

    摘要翻译： 本发明涉及从赭曲霉孢子的mRNA产生的cDNA文库中分离的新型细胞色素P450样酶（赭曲霉11α羟化酶）和氧化还原酶（赭曲霉氧化还原酶）。 当编码11α羟化酶的cDNA在草地贪夜蛾（Sf-9）昆虫细胞中以编码人氧化还原酶的cDNA作为电子给体共同表达时，其成功地催化类固醇底物4-雄甾烯-3,17- （AD）至11α-羟基-AD，通过HPLC分析测定。 本发明还涉及与这些cDNA相关或衍生的核酸分子，包括其互补序列，同系物和片段，以及使用这些核酸分子的方法，以产生例如其多肽及其片段。 本发明还涉及识别赭曲霉11α羟化酶和氧化还原酶的抗体的产生以及使用这些抗体分别在未修饰和转化的宿主细胞中检测这些天然和重组多肽的存在的方法。 本发明还提供在异源宿主细胞中单独或与人或曲霉属氧化还原酶组合表达曲霉11α羟化酶基因的方法，以促进类固醇底物对其11α羟基对应物的生物转化。

    Aspergillus ochraceus 11 alpha hydroxylase and oxidoreductase
    2.
    发明授权
    Aspergillus ochraceus 11 alpha hydroxylase and oxidoreductase 失效
    曲霉曲霉11α羟化酶和氧化还原酶

    公开(公告)号：US07238507B2

    公开(公告)日：2007-07-03

    申请号：US10900856

    申请日：2004-07-28

    申请人： Suzanne L. Bolten ,  Robert A. Clayton ,  Alan M. Easton ,  Leslie C. Engel ,  Dean M. Messing ,  John S. Ng ,  Beverly Reitz ,  Mark C. Walker ,  Ping T. Wang

    发明人： Suzanne L. Bolten ,  Robert A. Clayton ,  Alan M. Easton ,  Leslie C. Engel ,  Dean M. Messing ,  John S. Ng ,  Beverly Reitz ,  Mark C. Walker ,  Ping T. Wang

    IPC分类号： C12N9/02

    CPC分类号： C12N9/0083 ,  C07K14/38 ,  C07K16/40 ,  C12N1/14 ,  C12N3/00 ,  C12N9/0004 ,  C12N2799/026 ,  C12P33/10

    摘要： The present invention relates to a novel cytochrome P450-like enzyme (Aspergillus ochraceus 11 alpha hydroxylase) and an oxidoreductase (Aspergillus ochraceus oxidoreductase) isolated from cDNA library generated from the mRNA of Aspergillus ochraceus spores. When the cDNA encoding the 11 alpha hydroxylase was co-expressed in Spodoptera frugiperda (Sf-9) insect cells with the cDNA encoding human oxidoreductase as an electron donor, it successfully catalyzed the conversion of the steroid substrate 4-androstene-3,17-dione (AD) to 11 alpha-hydroxy-AD as determined by HPLC analysis. The invention also relates to nucleic acid molecules associated with or derived from these cDNAs including complements, homologues and fragments thereof, and methods of using these nucleic acid molecules, to generate, for example, polypeptides and fragments thereof. The invention also relates to the generation of antibodies that recognizes the A. ochraceus 11 alpha hydroxylase and oxidoreductase and methods of using these antibodies to detect the presence of these native and recombinant polypeptides within unmodified and transformed host cells, respectively. The invention also provides methods of expressing the Aspergillus 11 alpha hydroxylase gene separately, or in combination with human or Aspergillus oxidoreductase, in heterologous host cells, to facilitate the bioconversion of steroid substrates to their 11 alpha hydroxy-counterparts.

    摘要翻译： 本发明涉及从赭曲霉孢子的mRNA产生的cDNA文库中分离的新型细胞色素P450样酶（赭曲霉11α羟化酶）和氧化还原酶（赭曲霉氧化还原酶）。 当编码11α羟化酶的cDNA在草地贪夜蛾（Sf-9）昆虫细胞中以编码人氧化还原酶的cDNA作为电子给体共同表达时，其成功地催化类固醇底物4-雄甾烯-3,17- （AD）至11α-羟基-AD，通过HPLC分析测定。 本发明还涉及与这些cDNA相关或衍生的核酸分子，包括其互补序列，同系物和片段，以及使用这些核酸分子的方法，以产生例如其多肽及其片段。 本发明还涉及识别赭曲霉11α羟化酶和氧化还原酶的抗体的产生以及使用这些抗体分别在未修饰和转化的宿主细胞中检测这些天然和重组多肽的存在的方法。 本发明还提供在异源宿主细胞中单独或与人或曲霉属氧化还原酶组合表达曲霉11α羟化酶基因的方法，以促进类固醇底物对其11α羟基对应物的生物转化。