Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Miller School of Medicine, Miami, Florida 33136, USA.
J Mol Recognit. 2010 Jan-Feb;23(1):74-83. doi: 10.1002/jmr.983.
Mutation in the tubby gene causes adult-onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby-like protein 1 (Tulp1), whose C-terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N-terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N-terminus (tubby-N) as bait to identify unknown binding proteins with open-reading-frame (ORF) phage display. T7 phage display was engineered with three improvements: high-quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait-binding proteins in as fast as approximately 4-7 days. While phage display with conventional cDNA libraries identifies high percentage of out-of-frame unnatural short peptides, all 28 tubby-N-binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two-hybrid assay and protein pull-down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby-specific binding protein. These data suggest that tubby-N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly-engineered ORF phage display is a powerful technology to identify unknown protein-protein interactions.
突变的 tubby 基因导致成人发病肥胖、进行性视网膜和耳蜗退化，但其机制未知。相比之下，tubby-like protein 1（Tulp1）基因突变仅导致视网膜退化，而 Tulp1 的 C 末端与 tubby 高度同源。我们推测它们不同的 N 末端可能决定了它们不同的疾病特征。为了阐明 tubby 的结合伴侣，我们使用 tubby N 末端（tubby-N）作为诱饵，利用开放阅读框（ORF）噬菌体展示来鉴定未知的结合蛋白。T7 噬菌体展示经过了三个改进：高质量的 ORF 噬菌体展示 cDNA 文库、通过蛋白酶切割特异性洗脱噬菌体以及双噬菌体展示用于灵敏的高通量筛选。该新系统能够在大约 4-7 天内快速识别未知诱饵结合蛋白。虽然使用常规 cDNA 文库的噬菌体展示可以识别出高比例的非框架天然短肽，但 ORF 噬菌体展示鉴定的 28 个 tubby-N 结合克隆均为 ORF。它们编码 16 种蛋白质，包括 8 种核蛋白。用酵母双杂交和蛋白 pull-down 实验分析了 14 种蛋白质，其中 10 种蛋白被独立验证。比较结合分析揭示了几种与 tubby 和 Tulp1 都结合的蛋白质，以及一种 tubby 特异性结合蛋白。这些数据表明 tubby-N 能够与多种核内和细胞质蛋白结合伴侣相互作用。这些结果表明，新设计的 ORF 噬菌体展示是一种强大的技术，可以识别未知的蛋白质-蛋白质相互作用。
