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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Versatile Selection Technology For Intracellular Protein-protein Interactions Mediated By A Unique Bacterial Hitchhiker Transport Mechanism
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Versatile Selection Technology For Intracellular Protein-protein Interactions Mediated By A Unique Bacterial Hitchhiker Transport Mechanism

机译：独特的细菌搭便车转运机制介导的细胞内蛋白质相互作用的多功能选择技术

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摘要

We have developed a reliable genetic selection strategy for isolating interacting proteins based on the "hitchhiker" mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway. This method, designated FLI-TRAP (functional ligand-binding identification by Tat-based recognition of associating proteins), is based on the unique ability of the Tat system to efficiently cotranslocate nonco-valent complexes of 2 folded polypeptides. In the FLI-TRAP assay, the protein to be screened for interactions is engineered with an N-terminal Tat signal peptide, whereas the known or putative partner protein is fused to mature TEM-1 β-lactamase (Bla). Using a series of c-Jun and c-Fos leucine zipper (JunLZ and FosLZ) variants of known affinities, we observed that only those chimeras that expressed well and interacted strongly in the cytoplasm were able to colocalize Bla into the periplasm and confer β-lactam antibiotic resistance to cells. Likewise, the assay was able to efficiently detect interactions between intracellular single-chain Fv (scFv) antibodies and their cognate antigens. The utility of FLI-TRAP was then demonstrated through random library selections of amino acid substitutions that restored (i) heterodimerization to a noninteracting FosLZ variant, and (ii) antigen binding to a low-affinity scFv antibody. Because Tat substrates must be correctly folded before transport, FLI-TRAP favors the identification of soluble, nonaggregating, protease-resistant protein pairs and, thus, provides a powerful tool for routine selection of interacting partners (e.g., antibody-antigen), without the need for purification or immobilization of the binding target.
机译：我们已经开发了一种可靠的遗传选择策略，用于基于大肠杆菌双精氨酸易位（Tat）途径的“搭便车”机制来分离相互作用的蛋白质。这种方法称为FLI-TRAP（通过基于Tat的关联蛋白识别识别功能性配体结合），是基于Tat系统有效共转位2个折叠多肽的非共价复合物的独特能力。在FLI-TRAP分析中，要筛选相互作用的蛋白质是用N末端Tat信号肽改造的，而已知的或推定的伴侣蛋白质则与成熟的TEM-1β-内酰胺酶（Bla）融合。使用一系列已知亲和力的c-Jun和c-Fos亮氨酸拉链变体（JunLZ和FosLZ），我们观察到只有那些在细胞质中表达良好且相互作用强烈的嵌合体才能将Bla共定位于周质并赋予β-内酰胺抗生素对细胞的抵抗力。同样，该测定法能够有效地检测细胞内单链Fv（scFv）抗体与其同源抗原之间的相互作用。然后通过随机库选择氨基酸取代来证明FLI-TRAP的效用，该氨基酸取代可恢复（i）异二聚体化为非相互作用的FosLZ变体，和（ii）抗原与低亲和力scFv抗体的结合。由于Tat底物在运输前必须正确折叠，因此FLI-TRAP有助于鉴定可溶性，非聚集性，耐蛋白酶的蛋白质对，从而为常规选择相互作用伴侣（例如抗体-抗原）提供了有力工具，而无需需要纯化或固定结合靶标。

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