Department of Chemical Science and Engineering, Graduate School of Engineering, and Organization of Advanced Science and Technology, Kobe University, Nada, Kobe 657-8501, Japan.
Bioconjug Chem. 2010 Dec 15;21(12):2227-33. doi: 10.1021/bc100206z. Epub 2010 Nov 11.
A ZZ domain (ZZ) and alkaline phosphatase (AP), luciferase (Luc), or glucose oxidase (GOD) were conjugated using Sortase A (SrtA) from Staphylococcus aureus. The specific peptidyl linker for SrtA was genetically fused to the C-terminus of ZZ, and the other linker was fused to the N-terminus of AP, Luc, or GOD, respectively. The resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged ZZ and AP, Luc, or GOD were site-specifically conjugated by SrtA through the extra peptidyl linkers in vitro. The SrtA reaction had little influence on either the antibody-binding activity of the ZZ moiety or the enzymatic activity of AP, Luc, or GOD moieties of the conjugates. Additionally, antibody-ZZ-proteins were yielded easily by mixing antibody with ZZ-AP, ZZ-Luc, or ZZ-GOD, allowing their use in an enzyme-linked immunosorbent assay. These results suggest that the enzymatic approach with SrtA facilitates the construction of ZZ-proteins. Furthermore, mixing antibody and ZZ-proteins produces a wide variety of antibody-ZZ-proteins.
一个 ZZ 结构域（ZZ）和碱性磷酸酶（AP）、荧光素酶（Luc）或葡萄糖氧化酶（GOD）通过金黄色葡萄球菌的 Sortase A（SrtA）进行连接。SrtA 的特定肽连接子通过遗传融合到 ZZ 的 C 末端，而另一个连接子分别融合到 AP、Luc 或 GOD 的 N 末端。通过细菌表达分别获得了这些蛋白质。重组的肽标记的 ZZ 和 AP、Luc 或 GOD 通过 SrtA 在体外通过额外的肽连接子进行位点特异性连接。SrtA 反应对 ZZ 部分的抗体结合活性或连接物的 AP、Luc 或 GOD 部分的酶活性几乎没有影响。此外，通过将抗体与 ZZ-AP、ZZ-Luc 或 ZZ-GOD 混合，很容易产生抗体-ZZ 蛋白，这使其可用于酶联免疫吸附测定。这些结果表明，SrtA 的酶法有助于 ZZ 蛋白的构建。此外，混合抗体和 ZZ 蛋白可产生多种抗体-ZZ 蛋白。
