Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
Department of Applied Physics, Stanford University, Stanford, CA 94305, USA.
Mol Cell. 2019 Mar 7;73(5):1075-1082.e4. doi: 10.1016/j.molcel.2019.02.019.
High-throughput DNA sequencing techniques have enabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3 × 10 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a "superFLAG" epitope variant. We also measured the enzymatic activity of 1.56 × 10 molecular variants of full-length human O-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates revealed amino acid interaction networks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues.
高通量 DNA 测序技术为将 DNA 序列与生化功能联系起来提供了多种方法。相比之下，蛋白质功能的测定在通量、自动化和广泛可用性方面存在很大的局限性。我们已经将 Illumina 高通量测序芯片改编为展示核糖体翻译的蛋白质和肽的巨大多样性，然后直接在这个流动池上进行基于荧光的功能测定，证明了一个单一的、广泛可用的高通量平台可以同时进行合成测序和蛋白质测定。我们定量了 M2 抗 FLAG 抗体与 1.3×10 个 FLAG 肽变体文库的结合，探索了突变组合的非加性效应，并发现了一个“超级 FLAG”表位变体。我们还测量了全长人 O-烷基鸟嘌呤-DNA 烷基转移酶 (SNAP-tag) 的 1.56×10 个分子变体的酶活性。这个全面的催化速率文库揭示了氨基酸相互作用网络和协同性，将正协同性与结构接近性联系起来，并揭示了与组氨酸残基普遍存在的正协同性相互作用。
