Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario, Canada.
PLoS One. 2023 Apr 12;18(4):e0281228. doi: 10.1371/journal.pone.0281228. eCollection 2023.
Protein tandem repeats (TRs) are motifs comprised of near-identical contiguous sequence duplications. They are found in approximately 14% of all proteins and are implicated in diverse biological functions facilitating both structured and disordered protein-protein and protein-DNA interactions. These functionalities make protein TR domains an attractive component for the modular design of protein constructs. However, the repetitive nature of DNA sequences encoding TR motifs complicates their synthesis and mutagenesis by traditional molecular biology workflows commonly employed by protein engineers and synthetic biologists. To address this challenge, we developed a computational protocol to significantly reduce the complementarity of DNA sequences encoding TRs called TReSR (for Tandem Repeat DNA Sequence Redesign). The utility of TReSR was demonstrated by constructing a novel constitutive repressor synthesized by duplicating the LacI DNA binding domain into a single-chain TR construct by assembly PCR. Repressor function was evaluated by expression of a fluorescent reporter delivered on a single plasmid encoding a three-component genetic circuit. The successful application of TReSR to construct a novel TR-containing repressor with a DNA sequence that is amenable to PCR-based construction and manipulation will enable the incorporation of a wide range of TR-containing proteins for protein engineering and synthetic biology applications.
蛋白质串联重复（TR）是由近相同连续序列重复组成的基序。它们存在于大约 14%的所有蛋白质中，并涉及多种生物学功能，促进了结构和无序的蛋白质-蛋白质和蛋白质-DNA 相互作用。这些功能使蛋白质 TR 结构域成为蛋白质构建体模块化设计的有吸引力的组成部分。然而，编码 TR 基序的 DNA 序列的重复性质使它们的合成和诱变变得复杂，这是蛋白质工程师和合成生物学家通常采用的传统分子生物学工作流程。为了解决这个挑战，我们开发了一种计算协议，称为 TReSR（用于串联重复 DNA 序列重新设计），可以显着降低编码 TR 的 DNA 序列的互补性。TReSR 的实用性通过构建由单个链 TR 构建体通过组装 PCR 重复 LacI DNA 结合结构域而合成的新型组成型抑制剂来证明。通过在单个质粒上表达一个三组分遗传回路来递送荧光报告基因来评估抑制剂的功能，该质粒编码一个三组分遗传回路。TReSR 成功应用于构建一种新型的 TR 含有抑制剂，其 DNA 序列可通过基于 PCR 的构建和操作进行处理，这将使包含广泛的 TR 含有蛋白质的蛋白质工程和合成生物学应用成为可能。
