Abstract

The MAP tag system comprises a 14-residue peptide derived from mouse podoplanin and its high-affinity monoclonal antibody PMab-1. We determined the crystal structure of PMab-1 complexed with the MAP tag peptide and found that the recognition required only the N-terminal 8 residues of MAP tag sequence, enabling the shortening of the tag length without losing the affinity for PMab-1. Furthermore, the structure illustrated that the MAP tag adopts a U-shaped conformation when bound by PMab-1, suggesting that loop-inserted MAP tag would assume conformation compatible with the PMab-1 binding. We inserted the 8-residue MAP tag into multiple loop regions in various proteins including fibronectin type III domain and G-protein-coupled receptors and tested if they maintain PMab-1 reactivity. Despite the conformational restraints forced by the insertion position, all MAP-inserted mutants were expressed well in mammalian cells at levels comparable to the non-tagged proteins. Furthermore, the binding by PMab-1 was fully maintained even for the mutant where MAP tag was inserted at a structurally restricted β-hairpin, indicating that the MAP tag system has unique feature that allows placement in the middle of protein domain at desired locations. Our results indicate the versatile utility of the MAP tag system in ‘site-specific epitope insertion’ application.

中文翻译：

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使用MAP标签系统将位点特异性表位插入重组蛋白。

摘要

MAP标签系统包含14个残基的肽，该肽衍生自小鼠Podoplanin及其高亲和力单克隆抗体PMab-1。我们确定了与MAP标签肽复合的PMab-1的晶体结构，发现该识别仅需要MAP标签序列的N端8个残基，从而能够缩短标签长度而不会失去对PMab-1的亲和力。此外，该结构说明当与PMab-1结合时MAP标签采用U形构象，这表明插入环的MAP标签将假定与PMab-1结合兼容的构象。我们将8位残基的MAP标签插入到包括纤连蛋白III型结构域和G蛋白偶联受体在内的各种蛋白质的多个环区域中，并测试了它们是否维持PMab-1反应性。尽管受到插入位置的构象限制，但所有插入MAP的突变体在哺乳动物细胞中的表达水平均与未标记蛋白相当。此外，即使对于在结构上受限制的β-发夹处插入了MAP标签的突变体，也完全维持了PMab-1的结合，这表明MAP标签系统具有独特的功能，可以将其放置在蛋白质结构域的中间位置。我们的结果表明MAP标签系统在“特定于位点的表位插入”应用中具有广泛的用途。表示MAP标签系统具有独特的功能，可以将其放置在蛋白质结构域的中间所需位置。我们的结果表明MAP标签系统在“特定于位点的表位插入”应用中具有广泛的用途。表示MAP标签系统具有独特的功能，可以将其放置在蛋白质结构域的中间所需位置。我们的结果表明MAP标签系统在“特定于位点的表位插入”应用中具有广泛的用途。