Optimizing recombinant antibodies for intracellular function using hitchhiker-mediated survival selection.
Optimizing recombinant antibodies for intracellular function using hitchhiker-mediated survival selection.
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DOI：
10.1093/protein/gzu038
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发表时间：
2014-10
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影响因子：
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通讯作者：
D. Waraho-Zhmayev;Bunyarit Meksiriporn;Alyse D. Portnoff;M. DeLisa
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文献类型：
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作者：
D. Waraho-Zhmayev;Bunyarit Meksiriporn;Alyse D. Portnoff;M. DeLisa
细菌双精氨酸易位途径的“搭便车”机制先前已被改编为用于检测活大肠杆菌细胞的细胞质中的成对蛋白质相互作用的遗传选择。在这里，我们扩展了这种称为FLI-TRAP的方法，用于快速分离单链Fv形式的细胞内抗体（胞内抗体），这些抗体仅通过要求细菌在高浓度抗生素上生长就具有上级特性。在仅使用FLI-TRAP进行单轮基于存活的富集后，分离了针对酵母Gcn 4p转录因子的二聚化结构域的胞内抗体的变体，其具有显著更大的细胞内稳定性，其翻译产生>10倍的增强。同样，分离出对α-突触核蛋白的非淀粉样蛋白组分区域具有特异性的胞内抗体，其具有约8倍的提高的抗原结合亲和力。总的来说，我们的结果说明了FLI-TRAP方法用于胞内抗体的胞内稳定和亲和力成熟的潜力，所有这些都不需要纯化或固定抗原。
The 'hitchhiker' mechanism of the bacterial twin-arginine translocation pathway has previously been adapted as a genetic selection for detecting pairwise protein interactions in the cytoplasm of living Escherichia coli cells. Here, we extended this method, called FLI-TRAP, for rapid isolation of intracellular antibodies (intrabodies) in the single-chain Fv format that possess superior traits simply by demanding bacterial growth on high concentrations of antibiotic. Following just a single round of survival-based enrichment using FLI-TRAP, variants of an intrabody against the dimerization domain of the yeast Gcn4p transcription factor were isolated having significantly greater intracellular stability that translated to yield enhancements of >10-fold. Likewise, an intrabody specific for the non-amyloid component region of α-synuclein was isolated that has ~8-fold improved antigen-binding affinity. Collectively, our results illustrate the potential of the FLI-TRAP method for intracellular stabilization and affinity maturation of intrabodies, all without the need for purification or immobilization of the antigen.