用于结合物选择的膜蛋白生物素化。

Biotinylation of Membrane Proteins for Binder Selections.

机构信息

Institute for Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt, Germany.

Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.

出版信息

Methods Mol Biol. 2020;2127:151-165. doi: 10.1007/978-1-0716-0373-4_11.

DOI:10.1007/978-1-0716-0373-4_11

PMID:32112321

Abstract

The selective immobilization of proteins represents an essential step in the selection of binding proteins such as antibodies. The immobilization strategy determines how the target protein is presented to the binders and thereby directly affects the experimental outcome. This poses specific challenges for membrane proteins due to their inherent lack of stability and limited exposed hydrophilic surfaces. Here we detail methodologies for the selective immobilization of membrane proteins based on the strong biotin-avidin interaction and with a specific focus on its application for the selection of nanobodies and sybodies. We discuss the challenges in generating and benefits of obtaining an equimolar biotin to target-protein ratio.

摘要

蛋白质的选择性固定化是筛选结合蛋白（如抗体）的重要步骤。固定化策略决定了靶蛋白向结合物的呈现方式，从而直接影响实验结果。由于膜蛋白固有的不稳定性和有限的暴露亲水面，这给它们带来了特殊的挑战。在这里，我们详细介绍了基于强生物素-亲和素相互作用的膜蛋白选择性固定化方法，并特别关注其在纳米抗体和 sybodies 筛选中的应用。我们讨论了生成等摩尔生物素与靶蛋白比的挑战和益处。