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    • 7. 发明公开
    • METHOD FOR REFINING PROTEIN INCLUDING SELF-CUTTING CASSETTE AND USE THEREOF
    • 方法精制蛋白自切割带及其用途
    • EP2990423A1
    • 2016-03-02
    • EP14787873.0
    • 2014-04-25
    • Scripps Korea Antibody Institute
    • SONG, Byeong DooYUN, Jee SunCHOI, Hyo JungKIM, Hye InLEE, Eung-Suk
    • C07K19/00C12N15/62C12N15/70C07K1/16
    • C07K16/00C07K1/22C07K2319/00C07K2319/21C07K2319/30C07K2319/41C07K2319/42C07K2319/50C12N9/50C12N9/52C12N15/1093C12Y304/2207
    • The present invention relates to a self-cutting fusion protein including sequentially a target protein, a peptide consisting of an amino acid sequence of LPXTG, a sortase A cutting function domain, and a tag from the amino terminal; a coding nucleic acid thereof; an expression vector of the present invention including a nucleic acid; and a cell transformed by the expression vector of the present invention. The present invention also relates to a target protein refinement method including the steps of culturing, dissolving, and refining the transformed cell. The present invention relates to a self-cutting fusion protein including a self-cutting cassette which consists of a sortase A cutting function domain and a peptide consisting of an LPXTG amino acid sequence that is a recognition sequence thereof, and is very useful in that the steps of refining a target protein and eliminating the tag thereof can be accomplished by a single refinement step instead of separate steps. Particularly, the invention may be employed in various fields requiring a huge amount of a protein of a high purity in that a target protein existing in an amino terminal enhances the column union ability and the self-cutting ability of a fusion protein, a target protein rid of the tag is obtained with a high purity, a cutting-buffer makes the steps of refining and eliminating the tag be accomplished through a single step, significantly reducing the time and efforts needed for refining, and the accomplishment through a single step reduces the loss of the protein yield. Particularly, the invention is useful for manufacturing a therapeutic antibody-drug complex.
    • 本发明涉及一种自攻融合蛋白,依次包括靶蛋白,在LPXTG,一个分选酶A切割功能域组成的肽的氨基酸序列,并且从氨基末端标签; 一个编码它们的核酸; 本发明的包括一个核酸的表达载体的; 和细胞通过本发明的表达载体转化。 因此,本发明涉及一种靶蛋白的改进方法,其包括培养的步骤,溶解,并改进转化的细胞。 本发明涉及一种自攻融合蛋白包括自切割盒,其besteht分选酶的切割功能结构域和LPXTG氨基酸序列组成并的肽是其识别序列,并且是在DASS模具非常有用 炼目标蛋白和消除标签它们的步骤可以由一个单一的细化步骤,而不是单独的步骤中完成。 特别是,本发明可以在各种领域中,需要巨大的高纯度的蛋白质的量,DASS在氨基末端存在于靶蛋白可以采用增强列联合能力和融合蛋白的自切割能力,靶蛋白 去掉标签获得具有高纯度的,切割缓冲器使得精炼和消除标签的步骤,通过一个单一的步骤中完成,显着地减少所需的用于精炼的时间和努力,并通过一个单一的步骤中完成降低了 蛋白质产量的损失。 具体地说,本发明是用于制造治疗性抗体 - 药物复合物是有用的。