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    NOVEL METHODS FOR THE STABILISATION OF IMMUNOGLOBULIN CONSTANT DOMAINS
    3.
    发明申请
    NOVEL METHODS FOR THE STABILISATION OF IMMUNOGLOBULIN CONSTANT DOMAINS 审中-公开

    公开(公告)号：US20160340412A1

    公开(公告)日：2016-11-24

    申请号：US15110097

    申请日：2015-01-07

    Applicant: TECHNISCHE UNIVERSITÄT MÜNCHEN ,  HELMHOLTZ ZENTRUM MÜNCHEN DEUTSCHES FORSCHUNGSZENTRUM FÜR GESUNDHEIT UND UMWELT (GMBH)

    Inventor： Johannes Buchner ,  Matthias Feige ,  Moritz Marcinowski ,  Janosch Hennig

    IPC: C07K16/00

    CPC classification number: C07K16/00 ,  C07K2317/21 ,  C07K2317/522 ,  C07K2317/524 ,  C07K2317/526 ,  C07K2317/528 ,  C07K2317/94

    Abstract: The present invention relates to a method of producing a modified protein having an increased stability and/or improved folding efficiency as compared to the unmodified protein, the method comprising (i) modifying a nucleic acid molecule encoding a protein comprising at least one immunoglobulin constant domain-like region by (a-i) replacing the nucleotides encoding at least one amino acid, preferably an uncharged amino acid, in the loop separating the C strand from the D strand with nucleotides encoding a charged amino acid selected from the group consisting of Arg, Lys, His, Glu and Asp and/or replacing the nucleotides encoding at least one amino acid, preferably an uncharged amino acid, in the helix connecting the E strand with the F strand with nucleotides encoding a charged amino acid selected from the group consisting of Arg, Lys, His, Glu and Asp; (a-ii) replacing the nucleotides encoding at least one amino acid not having a side chain that can form a hydrogen bond in the loop separating the C strand from the D strand with nucleotides encoding an amino acid having a side chain capable of forming a hydrogen bond selected from the group consisting of Gln, Asn, Tyr, Ser and Thr and/or replacing the nucleotides encoding at least one amino acid not having a side chain that can form a hydrogen bond in the helix connecting the E strand with the F strand with nucleotides encoding an amino acid having a side chain capable of forming a hydrogen bond selected from the group consisting of Gln, Asn, Tyr, Ser and Thr; and/or (a-iii) replacing the nucleotides encoding at least one amino acid in the loop separating the C strand from the D strand with nucleotides encoding a cysteine and/or replacing the nucleotides encoding at least one amino acid in the helix connecting the E strand with the F strand with nucleotides encoding a cysteine; and/or (b) replacing the nucleotides encoding at least one non-hydrophobic amino acid at a position suitable to participate in the formation of the hydrophobic core with nucleotides encoding a hydrophobic amino acid selected from the group consisting of Val, Ile, Leu, Met, Phe, Trp and Pro; and (ii) expressing the nucleic acid molecule to produce the stabilised protein. The present invention further relates to a method of producing a modified protein having an improved secretion from cells as compared to the unmodified protein, as well as to a protein comprising at least one immunoglobulin constant domain-like region having an additional salt bridge, an additional hydrogen bond, an additional disulfide bridge and/or an extended hydrophobic core. The present invention further relates to a nucleic acid molecule encoding the modified protein of the invention, as well as a vector comprising said nucleic acid molecule and a host cell comprising the vector. Further, the present invention relates also to a composition as well as to a kit.

    Protein-based assays for screening of the IgE-receptor interaction
    5.
    发明授权
    Protein-based assays for screening of the IgE-receptor interaction 有权
    用于筛选IgE受体相互作用的基于蛋白质的测定法

    公开(公告)号：US08927695B2

    公开(公告)日：2015-01-06

    申请号：US13074543

    申请日：2011-03-29

    Applicant: Theodore S Jardetzky ,  Beomkyu Kim

    Inventor： Theodore S Jardetzky ,  Beomkyu Kim

    IPC: C07K16/00 ,  C07K16/42

    CPC classification number: C07K16/4291 ,  C07K16/00 ,  C07K2317/526 ,  C07K2317/528 ,  G01N2500/02 ,  Y10S530/862 ,  Y10S530/866

    Abstract: Embodiments of the invention are related to a polypeptide comprising the amino acid sequence of a human IgE-Fc Cε3-Cε4, wherein said Cε3-Cε4 starts at amino acid 328 and ends at amino acid 547 of said IgE-Fc, and wherein C 328 is A and K 367 is C. Other embodiments concern a second polypeptide comprising the amino acid sequence of a human FcεRIα extracellular region, wherein said extracellular region starts at amino acid 1 and ends at amino acid 176 of said FcεRIα. Still other embodiments are related to a method of identifying a compound that inhibits the binding of an IgE-Fc to a FcεRIα, said method comprising: contacting the polypeptide, wherein said IgE-Fc Cε3-Cε4 sequence is labeled with a fluorophore, and the second polypeptide, with a test compound; and determining whether binding of said polypeptide to said second polypeptide is decreased in the presence of said test compound.

    Abstract translation: 本发明的实施方案涉及包含人IgE-Fc C 3'和C 3的氨基酸序列的多肽，其中所述C 3和C 4的氨基酸序列从氨基酸328开始，并在所述 IgE-Fc，其中C 328为A且K 367为C.其他实施方案涉及包含人FcγRI胞外区的氨基酸序列的第二多肽，其中所述胞外区起始于氨基酸1并以氨基酸结束 176的所述FcγRIα。 还有其它实施方案涉及鉴定抑制IgE-Fc与FcγRIα结合的化合物的方法，所述方法包括：使所述多肽接触，其中所述IgE-Fc C 3 -C 8序列被标记 用荧光团和第二多肽与测试化合物反应; 以及在所述测试化合物的存在下确定所述多肽与所述第二多肽的结合是否降低。

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