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    • 3. 发明授权
    • Compositions and methods for immunoaffinity purification
    • 免疫亲和纯化的组合物和方法
    • US07534861B2
    • 2009-05-19
    • US10340179
    • 2003-01-10
    • Kenneth J. RothschildSadanand GiteJerzy OlejnikMark Lim
    • Kenneth J. RothschildSadanand GiteJerzy OlejnikMark Lim
    • C07K14/00C12N15/00
    • C07K14/4746C07K2319/20C07K2319/50C07K2319/70
    • A hybrid polypeptide composed of a p53 epitope peptide and a desired functional protein are produced by recombinant DNA techniques. A DNA expression vector is constructed that includes segments of DNA coding for the epitope peptide and the desired functional protein. An optional linking portion is contemplated. The linking portion of the epitope peptide is cleavable at a specific amino acid residue adjacent the functional protein by use of a sequence specific proteolytic enzyme or chemical proteolytic agent. The hybrid polypeptide expressed by the host cells transformed by the cloning vector is removed therefrom and purified by affinity chromatography techniques by use of an immobilized antibody specific to the antigenic portion of the epitope peptide. The protein is then cleaved from the isolated hybrid polypeptide with an appropriate proteolic enzyme or chemical agent, thereby releasing the mature functional protein in highly purified, highly active state.
    • 通过重组DNA技术产生由p53表位肽和所需功能性蛋白质组成的杂合多肽。 构建DNA表达载体,其包括编码表位肽和所需功能性蛋白质的DNA片段。 预期可选的连接部分。 表位肽的连接部分可以通过使用序列特异性蛋白水解酶或化学蛋白水解剂在与功能蛋白相邻的特定氨基酸残基处可切割。 由克隆载体转化的宿主细胞表达的杂合多肽从其中除去,并通过使用对表位肽的抗原部分特异性的固定化抗体通过亲和层析技术进行纯化。 然后用合适的蛋白酶或化学试剂从分离的杂交多肽切割蛋白质,从而释放高度纯化,高活性状态的成熟功能性蛋白质。