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    • 2. 发明申请
    • t-PA MUTANT GK1L
    • WO1991002798A2
    • 1991-03-07
    • PCT/EP1990001401
    • 1990-08-22
    • BOEHRINGER MANNHEIM GMBHWEIDLE, UlrichSTERN, Anne
    • BOEHRINGER MANNHEIM GMBH
    • C12N15/58
    • C12N9/6459A61K38/00C12Y304/21069
    • 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 in the context of the genetic code are completely deleted in accordance with the exact exon/intron limits on the t-PA gene. Process for preparing said recombinant DNA. Vectors which contain said recombinant DNA as well as cells which are transformed with said vectors or said recombinant DNA. Protein with fibrinolytic properties by expression of a DNA sequence according to the invention in suitable host cells, which is composed of the amino acid sequences of the domains G, K1 and L of t-PA in this sequence and is possibly glycosylized. Process for preparing it. Fibrinolytic agent containing a protein according to the invention.
    • 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的序列或其在遗传背景下衍生的序列 根据t-PA基因的确切外显子/内含子限制,代码完全被删除。 制备所述重组DNA的方法。 含有所述重组DNA的载体以及用所述载体或所述重组DNA转化的细胞。 通过在合适的宿主细胞中表达根据本发明的DNA序列的纤维蛋白溶解性质的蛋白质,其由该序列中t-PA的结构域G,K1和L的结构域G,K1和L的氨基酸序列组成,并且可能被糖基化。 准备过程 含有本发明蛋白质的纤维蛋白溶解剂。
    • 6. 发明申请
    • PROTEIN EXPRESSION FROM MULTIPLE NUCLEIC ACIDS
    • 来自多种核酸的蛋白质表达
    • WO2009046978A1
    • 2009-04-16
    • PCT/EP2008/008523
    • 2008-10-09
    • F. HOFFMANN-LA ROCHE AGGOEPFERT, UlrichKNOETGEN, HendrikKOPETZKI, ErhardSTERN, Anne
    • GOEPFERT, UlrichKNOETGEN, HendrikKOPETZKI, ErhardSTERN, Anne
    • C12N15/69C12N15/85C07K16/00
    • C12N15/85C07K16/18C07K16/28C07K16/2812C07K16/2854C07K16/2866C07K16/2896C07K2317/14C07K2317/21C07K2317/51C07K2317/515C12P21/00
    • 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.
    • 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,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。
    • 9. 发明申请
    • MUTANT OF THE ERYTHRINA CAFFRA TYPE INHIBITOR AND THE USE OF THE SAID MUTANT FOR PURIFYING SERIN PROTEASES
    • 刺桐caffra型抑制剂的突变体及其使用丝氨酸蛋白酶的清洁
    • WO1996031606A1
    • 1996-10-10
    • PCT/EP1996001388
    • 1996-03-29
    • BOEHRINGER MANNHEIM GMBHKOHNERT, UlrichSTERN, AnneWOZNY, ManfredFISCHER, Stephan
    • BOEHRINGER MANNHEIM GMBH
    • C12N15/15
    • C12N9/6459C07K14/8114C12Y304/21069
    • The invention concerns a polypeptide which has the activity of an inhibitor DE-3 from Erythrina caffra and reversibly and selectively binds serin proteases from a protein mixture, obtained by culturing prokaryotic or eukaryotic host cells transformed or transfected with a nucleic acid which codes for the polypeptide in question in such a way that the host cells, under favourable nutrient conditions, can express the polypeptide. The invention also concerns the isolation of the polypeptide. The polypeptide is characterised by having an amino acid sequence which is functionally analogous to SEQ ID NO:2, is in one region over 85 % homologous with the region of amino acids 39-139 of that sequence, has two disulphide bridges and begins N-terminally with SEQ ID NO:4 or with a SEQ ID NO:4 extended N terminally by methionin, has a binding capacity for tissue plasminogen activators of 1.25 MU/ml or more. The polypeptide in question is especially suitable for use in purifying plasminogen activators such as tissue plasminogen activators (t-PA and derivatives).
    • 其具有从刺桐caffra抑制剂DE-3的活性和可逆地和选择性的丝氨酸蛋白酶的多肽从蛋白质混合物通过对所述的方式多肽培养用一种核酸转化的编码原核或真核宿主细胞结合,或 被转染,其允许宿主细胞以表达合适的营养条件下,所述多肽和分离所述多肽,其特征在于,所述多肽具有的氨基酸序列,其是功能上类似于SEQ ID NO:2,在超过的部分区域 85%的同源性的氨基酸这个序列的39-139的区域中,具有两个二硫键和N端与SEQ ID NO:4或具有甲硫氨酸N-末端延伸SEQ ID NO:4点的开始和1.25 MU的组织型纤溶酶原激活剂的结合能力 / ml和具有更多的,特别是用于清洁v 合适的纤溶酶原活化剂,如组织纤维蛋白溶酶原活化剂(t-PA和衍生物)。