Engineering antibody fitness and function using membrane-anchored display of correctly folded proteins.
Engineering antibody fitness and function using membrane-anchored display of correctly folded proteins.
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DOI：
10.1016/j.jmb.2011.12.021
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发表时间：
2012-02-10
影响因子：
5.6
通讯作者：
DeLisa, Matthew P.
中科院分区：
文献类型：
--
作者：
Karlsson, Amy J.;Lim, Hyung-Kwon;Xu, Hansen;Rocco, Mark A.;Bratkowski, Matthew A.;Ke, Ailong;DeLisa, Matthew P.
关键词：
细菌双精氨酸易位（达特）途径的标志是其输出折叠蛋白的能力。在这里，我们发现，过表达的达特底物蛋白形成两个不同的，长寿命的易位中间体，很容易检测到的免疫标记方法。早期易位中间体Ti-1的形成将N-和C-末端暴露于细胞质，不需要完整的达特易位酶、功能性达特信号肽或正确折叠的底物。相比之下，后来的易位中间体Ti-2的形成要特殊得多，它表现出N末端在细胞质中、C末端在周质中的双位拓扑结构，需要完整的易位酶、功能性信号肽和正确折叠的底物蛋白。直接检测Ti-2中间体的能力随后被开发用于一种新的蛋白质工程技术，称为MAD-TRAP（膜锚定显示相关蛋白的基于Tat的识别）。仅用两轮诱变和用MAD-TRAP筛选，同时提高了人单链抗体片段的细胞内折叠和抗原结合活性。这种方法对于文库筛选具有几个优点，包括独特的达特折叠质量控制机制的参与，其确保仅展示天然样蛋白，从而从筛选过程中消除折叠不良的序列。
A hallmark of the bacterial twin-arginine translocation (Tat) pathway is its ability to export folded proteins. Here, we discovered that overexpressed Tat substrate proteins form two distinct, long-lived translocation intermediates that are readily detected by immunolabeling methods. Formation of the early translocation intermediate, Ti-1, which exposes the N- and C-termini to the cytoplasm, did not require an intact Tat translocase, a functional Tat signal peptide, or a correctly folded substrate. In contrast, formation of the later translocation intermediate, Ti-2, which exhibits a bitopic topology with the N-terminus in the cytoplasm and C-terminus in the periplasm, was much more particular, requiring an intact translocase, a functional signal peptide, and a correctly folded substrate protein. The ability to directly detect Ti-2 intermediates was subsequently exploited for a new protein engineering technology called MAD-TRAP (membrane-anchored display for Tat-based recognition of associating proteins). Using just two rounds of mutagenesis and screening with MAD-TRAP, the intracellular folding and antigen-binding activity of a human single-chain antibody fragment were simultaneously improved. This approach has several advantages for library screening, including the unique involvement of the Tat folding quality control mechanism that ensures only native-like proteins are displayed, thus eliminating poorly folded sequences from the screening process.
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影响因子：
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