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    Thrombolytically active non-glycosylated protein
    1.
    发明授权
    Thrombolytically active non-glycosylated protein 失效
    紫外线活性非糖蛋白

    公开(公告)号：US5223256A

    公开(公告)日：1993-06-29

    申请号：US585129

    申请日：1990-09-28

    申请人： Anne Stern ,  Ulrich Kohnert ,  Rainer Rudolph ,  Stephan Fischer ,  Ulrich Martin

    发明人： Anne Stern ,  Ulrich Kohnert ,  Rainer Rudolph ,  Stephan Fischer ,  Ulrich Martin

    IPC分类号： C12N15/09 ,  A61K38/00 ,  A61K38/46 ,  A61K38/48 ,  A61K38/49 ,  A61P7/02 ,  C07K1/113 ,  C07K14/745 ,  C12N9/64 ,  C12N9/72 ,  C12N9/88 ,  C12N15/58

    CPC分类号： C12N9/6459 ,  C07K1/1133 ,  C12Y304/21069 ,  A61K38/00

    摘要： A new thrombolytically active protein is not glycosylated and consists of the following amino acid sequence: ##STR1## or said amino acid sequence with an additional N-terminal methionine. DNA sequences encoding the thrombolytically active, non-glycosylated protein and pharmaceutical compositions containing said protein are also disclosed. The protein possesses particularly favorable properties when used to dissolve blood clots.

    摘要翻译： PCT No.PCT / EP90 / 00194 Sec。 371 1990年9月28日第 102（e）1990年9月28日PCT PCT 1990年2月6日PCT公布。 第WO90 / 09437号公报 日期8月23日，1990.A新血栓溶解活性蛋白未被糖基化，它包括以下氨基酸序列组成：1SYQGNSDCYFGNGSAYRGTHSLTESGASCL PWNSMILIGKVYTAQNPSAQ 51ALGLGKHNYCRNPDGDAKPWCHVLKNRRLT WEYCDVPSCSTCGLRQYSQP 101QFRIKGGLFADIASHPWQAAIFAKHRRSPG ERFLCGGILISSCWILSAAH 151CFQERFPPHHLTVILGRTYRVVPGEEEQKF EVEKYIVHKEFDDDTYDNDI 201ALLQLKSDSSRCAQESSVVRTVCLPPADLQ LPDWTECELSGYGKHEALSP 251FYSERLKEAHVRLYPSSRCTSQHLLNRTVT DNMLCAGDTRSGGPQANLHD 301ACQGDSGGPLVCLNDGRMTLVGIISWGLGC GQKDVPGVYTKVTNYLDWIR 351DNMRP或所述具有附加的N末端氨基酸序列 甲硫氨酸 还公开了编码血栓溶解活性的非糖基化蛋白质的DNA序列和含有所述蛋白质的药物组合物。 当用于溶解血块时，蛋白质具有特别有利的特性。

    Use of recombinant inhibitor from Erythrina caffra for purifying serine proteases
    4.
    发明授权
    Use of recombinant inhibitor from Erythrina caffra for purifying serine proteases 失效
    使用Erythrina caffra的重组抑制剂来纯化丝氨酸蛋白酶

    公开(公告)号：US5958722A

    公开(公告)日：1999-09-28

    申请号：US702703

    申请日：1996-09-13

    申请人： Ulrich Kohnert ,  Anne Stern ,  Stephan Fischer

    发明人： Ulrich Kohnert ,  Anne Stern ,  Stephan Fischer

    IPC分类号： C12N15/09 ,  C07K1/18 ,  C07K1/22 ,  C07K14/745 ,  C07K14/81 ,  C12N9/64 ,  C12N9/72 ,  C12N15/15 ,  C12P21/02 ,  C12R1/19 ,  C12P21/06 ,  C07H21/04 ,  C07K14/415 ,  C12N9/50

    CPC分类号： C12N9/6459 ,  C07K14/8114 ,  C12Y304/21069 ,  Y10S435/814 ,  Y10S435/815

    摘要： Process for purifying serine proteases from a protein mixture by binding the serine protease to an immobilized polypeptide with the activity of an inhibitor DE-3 from Erythrina caffra, removing unbound components from the protein mixture, detaching the serine protease from the inhibitor and separating the immobilized inhibitor from the soluble serine protease and isolating serine protease which is characterized in that a polypeptide is used as the polypeptide which is the product of a prokaryotic or eukaryotic expression of an exogenous nucleic acid. This inhibitor is distinguished by an improved specific activity and is particularly suitable for the purification of plasminogen activators such as tissue plasminogen activators (t-PA and derivatives).

    摘要翻译： PCT No.PCT / EP95 / 00926 Sec。 371日期1996年9月13日 102（e）1996年9月13日PCT PCT 1995年3月13日PCT公布。 公开号WO95 / 25168 日期1995年9月21日从蛋白质混合物中纯化丝氨酸蛋白酶的方法，通过将丝氨酸蛋白酶与来自Erythrina caffra的抑制剂DE-3的活性结合到固定化多肽上，从蛋白质混合物中除去未结合的成分，将丝氨酸蛋白酶从 将固定化的抑制剂与可溶性丝氨酸蛋白酶分离并分离丝氨酸蛋白酶，其特征在于使用多肽作为外源核酸的原核或真核表达产物的多肽。 该抑制剂的特征在于改善的比活性，特别适用于纤维蛋白溶酶原激活剂如组织纤溶酶原激活剂（t-PA和衍生物）的纯化。

    Mutant of the Erythrina caffra type inhibitor and the use of the said mutant for purifying serine proteases
    5.
    发明授权
    Mutant of the Erythrina caffra type inhibitor and the use of the said mutant for purifying serine proteases 失效
    Erythrina caffra型抑制剂的突变体和所述突变体用于纯化丝氨酸蛋白酶的用途

    公开(公告)号：US5973118A

    公开(公告)日：1999-10-26

    申请号：US943814

    申请日：1997-10-03

    申请人： Ulrich Kohnert ,  Anne Stern ,  Manfred Wozny ,  Stephan Fischer

    发明人： Ulrich Kohnert ,  Anne Stern ,  Manfred Wozny ,  Stephan Fischer

    IPC分类号： C12N15/09 ,  C07K1/22 ,  C07K14/81 ,  C12N1/21 ,  C12N9/64 ,  C12N9/72 ,  C12N9/99 ,  C12N15/29 ,  C12P21/02 ,  C12R1/19 ,  C07K1/00 ,  A61K35/78 ,  A61K38/00

    CPC分类号： C12N9/6459 ,  C07K14/8114 ,  C12Y304/21069

    摘要： A polypeptide which has the activity of an inhibitor DE-3 from Erythrina caffra and which reversibly and selectively binds serine proteases from a protein mixture is obtainable by culturing prokaryotic or eukaryotic host cells which have been transformed or transfected with a nucleic acid that codes for the said polypeptide in a manner that allows the host cells to express the said polypeptide under suitable nutrient conditions and isolating the said polypeptide, wherein the polypeptide has an amino acid sequence which is functionally analogous to SEQ ID NO:2, has a partial region that is more than 85% homologous to the region of amino acids 39-139 of this sequence, has two disulfide bridges and begins N-terminally with SEQ ID NO:4 or with a SEQ ID NO:4 extended N-terminally by methionine and has a binding capacity for tissue plasminogen activators of 1.25 MU/ml and more and is particularly suitable for purifying plasminogen activators such as tissue plasminogen activators (t-PA and derivatives).

    摘要翻译： 通过培养原核或真核宿主细胞获得具有来自艾瑞氏菌（Erythrina caffra）的抑制剂DE-3并且可逆地和选择性地结合蛋白质混合物的丝氨酸蛋白酶的多肽，所述原核或真核宿主细胞已经用编码 所述多肽以允许宿主细胞在合适的营养条件下表达所述多肽并分离所述多肽的方式，其中所述多肽具有功能上类似于SEQ ID NO：2的氨基酸序列，其部分区域是 与该序列的氨基酸39-139的区域同源超过85％具有两个二硫键，并与SEQ ID NO：4进行N-末端或与蛋氨酸N-末端扩增的SEQ ID NO：4，并具有 组织纤溶酶原激活剂的结合能力为1.25 MU / ml以上，特别适用于纯化纤溶酶原激活剂如组织纤溶酶原激活剂（t-PA和de rivatives）。

    PROTEIN EXPRESSION FROM MULTIPLE NUCLEIC ACIDS
    6.
    发明申请
    PROTEIN EXPRESSION FROM MULTIPLE NUCLEIC ACIDS 有权
    来自多种核酸的蛋白质表达

    公开(公告)号：US20100249379A1

    公开(公告)日：2010-09-30

    申请号：US12681781

    申请日：2008-10-09

    申请人： Ulrich Goepfert ,  Hendrik Knoetgen ,  Erhard Kopetzki ,  Anne Stern

    发明人： Ulrich Goepfert ,  Hendrik Knoetgen ,  Erhard Kopetzki ,  Anne Stern

    IPC分类号： C07K16/28 ,  C12P21/08

    CPC分类号： C12N15/85 ,  C07K16/18 ,  C07K16/28 ,  C07K16/2812 ,  C07K16/2854 ,  C07K16/2866 ,  C07K16/2896 ,  C07K2317/14 ,  C07K2317/21 ,  C07K2317/51 ,  C07K2317/515 ,  C12P21/00

    摘要： The current invention reports a method for the recombinant production of a secreted heterologous immunoglobulin in a CHO cell comprising the following steps: i) providing a CHO cell, which is adapted to growth in suspension culture, adapted to growth in serum-free medium, mycoplasma free, and virus free, ii) providing a vector comprising a prokaryotic origin of replication, a first nucleic acid conferring resistance to a prokaryotic selection agent, a second nucleic acid encoding the heavy chain of said heterologous immunoglobulin, a third nucleic acid encoding the light chain of said heterologous immunoglobulin, a fourth nucleic acid conferring resistance to a eukaryotic selection agent, iii) transfecting said CHO cell, wherein said transfecting comprises a) transfecting said CHO cell with said vector comprising a fourth nucleic acid conferring resistance to a first eukaryotic selection agent, b) selecting a CHO cell by growth in cultivation medium containing said first eukaryotic selection agent, c) transfecting said selected CHO cell with said vector comprising a fourth nucleic acid conferring resistance to a second eukaryotic selection agent different to said first eukaryotic selection agent, d) selecting a CHO cell by selected growth in cultivation medium containing said first and said second eukaryotic selection agent, iv) cultivating said transfected CHO cell in a medium in the presence of said first and second eukaryotic selection agent, under conditions suitable for the expression of said second, and third nucleic acid, and v) recovering said secreted heterologous immunoglobulin from the cultivation medium.

    摘要翻译： 本发明报告了在CHO细胞中重组产生分泌的异源免疫球蛋白的方法，其包括以下步骤：i）提供适于在悬浮培养物中生长的CHO细胞，适于在无血清培养基中生长，支原体 免费和无病毒，ii）提供包含原核复制起点的载体，赋予原核选择剂抗性的第一核酸，编码所述异源免疫球蛋白的重链的第二核酸，编码光的第三核酸 所述异源免疫球蛋白链，赋予对真核选择剂的抗性的第四核酸，iii）转染所述CHO细胞，其中所述转染包括a）用包含第四核酸的所述载体转染所述CHO细胞，所述第四核酸赋予对第一真核选择性的抗性 试剂，b）通过在含有所述第一种真核生物的培养基中生长来选择CHO细胞 c）用所述载体转染所述选择的CHO细胞，所述载体包含赋予与所述第一真核选择试剂不同的第二真核选择剂的抗性的第四核酸，d）通过在含有所述第一和第二真核选择试剂的培养基中选择生长来选择CHO细胞， 所述第二真核选择剂，iv）在所述第一和第二真核选择剂的存在下，在适于表达所述第二和第三核酸的条件下，在培养基中培养所述转染的CHO细胞，和v）回收所述分泌的异源 免疫球蛋白。

    Optimization of cells for endogenous gene activation
    8.
    发明授权
    Optimization of cells for endogenous gene activation 有权
    内源基因激活的细胞优化

    公开(公告)号：US07008764B1

    公开(公告)日：2006-03-07

    申请号：US09203500

    申请日：1998-12-01

    申请人： Konrad Honold ,  Thomas Holtschke ,  Anne Stern

    发明人： Konrad Honold ,  Thomas Holtschke ,  Anne Stern

    IPC分类号： C12Q1/68

    CPC分类号： C07K14/505 ,  C12N15/63 ,  C12N15/65 ,  C12N15/85 ,  C12N15/90 ,  C12N15/907 ,  C12N2800/30 ,  C12N2830/002 ,  C12Q1/6897

    摘要： The invention concerns a process for optimizing the gene expression in cells. A first aspect concerns a process for changing the expression of a nucleic acid sequence which is present endogenously in a eukaryotic cell by introduction of a heterologous expression control sequence into the genome of the cell by means of homologous recombination as well as site-specific recombinase-mediated excision of inserted foreign DNA and its replacement by further heterologous expression control sequences or/and amplification genes. In addition the invention concerns the introduction of one or several nucleic acid sequences to which an activator protein or an activator protein complex binds e.g. a hypoxia-inducible factor (HIF), into the genome of a eukaryotic cell by homologous recombination in order to change the expression of a target gene. Furthermore the invention concerns a process for testing the influence of 5′ or 3′ non-coding nucleic acid fragments on the expression of a target gene by determining the expression of a reporter gene. In addition the invention concerns a process for providing a DHFR-negative eukaryotic cell containing a recombinase target sequence as well as the expression of a nucleic acid sequence inserted into the recombinase target sequence.

    摘要翻译： 本发明涉及优化细胞中基因表达的方法。 第一方面涉及一种通过将异源表达控制序列通过同源重组以及位点特异性重组酶导入到细胞基因组中而在真核细胞中内源性存在的核酸序列的表达的方法。 介导的插入的外源DNA的切除及其通过进一步的异源表达控制序列或/和扩增基因的替代。 此外，本发明涉及一种或几种核酸序列的引入，活化蛋白或活化蛋白复合物例如与其结合。 缺氧诱导因子（HIF），通过同源重组进入真核细胞的基因组，以改变靶基因的表达。 此外，本发明涉及通过测定报告基因的表达来测试5'或3'非编码核酸片段对靶基因表达的影响的方法。 此外，本发明涉及提供含有重组酶靶序列的DHFR阴性真核细胞以及插入重组酶靶序列中的核酸序列的表达的方法。

    t-PA mutant GK1L
    9.
    发明授权
    t-PA mutant GK1L 失效
    T-PA MUTANT GK1L

    公开(公告)号：US5223422A

    公开(公告)日：1993-06-29

    申请号：US671776

    申请日：1991-04-16

    申请人： Ulrich Weidle ,  Anne Stern

    发明人： Ulrich Weidle ,  Anne Stern

    IPC分类号： C12N15/09 ,  A61K38/00 ,  A61K38/46 ,  A61P7/02 ,  C07K14/00 ,  C12N1/15 ,  C12N5/10 ,  C12N9/64 ,  C12N9/72 ,  C12N15/58 ,  C12R1/91

    CPC分类号： C12N9/6459 ,  C12Y304/21069 ,  A61K38/00

    摘要： The invention concerns a recombinant DNA which codes for a protein with the domains G, K1 and L of t-PA in which the sequences coding for the domains K2 and F of the wild-type t-PA gene or the sequences derived therefrom within the scope of the degeneration of the genetic code are completely deleted according to the exact exon/intron borders on the t-PA gene. The invention also relates to a process for the production of a recombinant DNA according to the present invention. In addition the invention concerns vectors containing this recombinant DNA as well as cells which are transformed with vectors according to the present invention or with the DNA according to the present invention. Furthermore the invention provides a protein with fibrinolytic properties by expression of a DNA sequence according to the present invention in suitable host cells which consists of the amino acid sequences of the domains G, K1 and L of t-PA in this order and which, if desired, is glycosylated as well as a process for its production. Finally the invention also concerns a fibrinolytic agent containing a protein according to the present invention.

    摘要翻译： PCT No.PCT / EP90 / 01401 Sec。 371日期1991年5月24日 102（e）日期1991年5月24日PCT提交1990年8月22日PCT公布。 出版物WO91 / 02798 1991年3月7日。本发明涉及编码具有t-PA结构域G，K1和L的蛋白质的重组DNA，其中编码野生型t-PA基因的结构域K2和F的序列 或者在遗传密码子的退化范围内衍生自其的序列根据t-PA基因上的确切的外显子/内含子边界被完全删除。 本发明还涉及根据本发明的重组DNA的制备方法。 此外，本发明涉及含有该重组DNA的载体以及根据本发明的载体或根据本发明的DNA转化的细胞。 此外，本发明提供了一种具有纤维蛋白溶解性质的蛋白质，其通过根据本发明的DNA序列在合适的宿主细胞中表达，所述合适的宿主细胞由t-PA的结构域G，K1和L的氨基酸序列依次组成， 所需的是糖基化的，以及其生产方法。 最后，本发明还涉及含有根据本发明的蛋白质的纤维蛋白溶解剂。

    Protein expression from multiple nucleic acids
    10.
    发明授权
    Protein expression from multiple nucleic acids 有权
    来自多个核酸的蛋白质表达

    公开(公告)号：US08771988B2

    公开(公告)日：2014-07-08

    申请号：US12681781

    申请日：2008-10-09

    申请人： Ulrich Goepfert ,  Hendrik Knoetgen ,  Erhard Kopetzki ,  Anne Stern

    发明人： Ulrich Goepfert ,  Hendrik Knoetgen ,  Erhard Kopetzki ,  Anne Stern

    IPC分类号： C12P21/08

    CPC分类号： C12N15/85 ,  C07K16/18 ,  C07K16/28 ,  C07K16/2812 ,  C07K16/2854 ,  C07K16/2866 ,  C07K16/2896 ,  C07K2317/14 ,  C07K2317/21 ,  C07K2317/51 ,  C07K2317/515 ,  C12P21/00

    摘要： The current invention reports a method for the recombinant production of a secreted heterologous immunoglobulin in a CHO cell comprising the following steps: i) providing a CHO cell, which is adapted to growth in suspension culture, adapted to growth in serum-free medium, mycoplasma free, and virus free, ii) providing a vector comprising a prokaryotic origin of replication, a first nucleic acid conferring resistance to a prokaryotic selection agent, a second nucleic acid encoding the heavy chain of said heterologous immunoglobulin, a third nucleic acid encoding the light chain of said heterologous immunoglobulin, a fourth nucleic acid conferring resistance to a eukaryotic selection agent, iii) transfecting said CHO cell, wherein said transfecting comprises a) transfecting said CHO cell with said vector comprising a fourth nucleic acid conferring resistance to a first eukaryotic selection agent, b) selecting a CHO cell by growth in cultivation medium containing said first eukaryotic selection agent, c) transfecting said selected CHO cell with said vector comprising a fourth nucleic acid conferring resistance to a second eukaryotic selection agent different to said first eukaryotic selection agent, d) selecting a CHO cell by selected growth in cultivation medium containing said first and said second eukaryotic selection agent, iv) cultivating said transfected CHO cell in a medium in the presence of said first and second eukaryotic selection agent, under conditions suitable for the expression of said second, and third nucleic acid, and v) recovering said secreted heterologous immunoglobulin from the cultivation medium.

    摘要翻译： 本发明报告了在CHO细胞中重组产生分泌的异源免疫球蛋白的方法，其包括以下步骤：i）提供适于在悬浮培养物中生长的CHO细胞，适于在无血清培养基中生长，支原体 免费和无病毒，ii）提供包含原核复制起点的载体，赋予原核选择剂抗性的第一核酸，编码所述异源免疫球蛋白的重链的第二核酸，编码光的第三核酸 所述异源免疫球蛋白链，赋予对真核选择剂的抗性的第四核酸，iii）转染所述CHO细胞，其中所述转染包括a）用包含第四核酸的所述载体转染所述CHO细胞，所述第四核酸赋予对第一真核选择性的抗性 试剂，b）通过在含有所述第一种真核生物的培养基中生长来选择CHO细胞 c）用所述载体转染所述选择的CHO细胞，所述载体包含赋予与所述第一真核选择剂不同的第二真核选择剂的抗性的第四核酸，d）通过在含有所述第一和第二真核选择剂的培养基中选择生长来选择CHO细胞， 所述第二真核选择剂，iv）在所述第一和第二真核选择剂的存在下，在适于表达所述第二和第三核酸的条件下，在培养基中培养所述转染的CHO细胞，和v）回收所述分泌的异源 免疫球蛋白。