Meyer Christoph, Liebscher Sandra, Bordusa Frank
Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg , Kurt-Mothes-Strasse 3, D-06120 Halle/Saale, Germany.
Bioconjug Chem. 2016 Jan 20;27(1):47-53. doi: 10.1021/acs.bioconjchem.5b00618. Epub 2015 Dec 21.
The combination of pure chemical methods with enzymatic approaches offers a kit system with maximum flexibility for site-specifically tagging proteins with a broad variety of artificial structures. Trypsiligase, a recently introduced designer enzyme for both N- and C-terminal site-specific labeling of peptides and proteins, has been used to introduce click anchors into the human protein cyclophilin 18 and the antibody Fab fragments anti-TNFα and anti-Her2. The subsequent click reactions with tetrazine or norbornene moieties lead to quantitative conversions to the corresponding dihydropyridazine products, thereby forming a stable covalent linkage between the label and the protein of interest. With this technology, cyclophilin 18 has been efficiently modified with the fluorescent dansyl moiety and the pharmaceutically relevant polymer PEG exclusively at its N-terminus. With the same methodology, the Fab fragments of anti-TNFα and anti-Her2 were derivatized exclusively at their C-terminal ends with PEG and the fluorescent dye carboxyfluorescein in the case of anti-TNFα or with the cytotoxic payload DM1 in the case of anti-Her2, to form a homogeneous antibody-drug conjugate (ADC).
将纯化学方法与酶促方法相结合，可提供一种试剂盒系统，该系统具有最大的灵活性，能够用多种人工结构对蛋白质进行位点特异性标记。胰蛋白酶连接酶是一种最近引入的用于肽和蛋白质N端和C端位点特异性标记的设计酶，已被用于将点击锚引入人亲环蛋白18以及抗TNFα和抗Her2抗体Fab片段中。随后与四嗪或降冰片烯部分的点击反应导致定量转化为相应的二氢哒嗪产物，从而在标签与目标蛋白质之间形成稳定的共价连接。利用这项技术，亲环蛋白18已仅在其N端用荧光丹磺酰部分和与药物相关的聚合物PEG进行了有效修饰。采用相同的方法，抗TNFα和抗Her2的Fab片段仅在其C端用PEG进行衍生化，抗TNFα的情况下用荧光染料羧基荧光素进行衍生化，抗Her2的情况下用细胞毒性载荷DM1进行衍生化，以形成均一的抗体-药物偶联物（ADC）。
