Recent years have witnessed increasing efforts to engineer artificial biological functions through recombination of modular-organized toolboxes of protein scaffolds and parts. A critical, yet frequently neglected aspect concerns the identity of peptide linkers or spacers connecting individual domains which remain poorly understood and challenging to assemble. Addressing these limitations, iFlinkC comprises a highly scalable DNA assembly process that facilitates the combinatorial recombination of functional domains with linkers of varying length and flexibility, thereby overcoming challenges with high GC-content and the repeat nature of linker elements. The capacity of iFLinkC is demonstrated in the construction of synthetic protease switches featuring PDZ-FN3-based affinity clamps and single-chain FKBP12-FRB receptors as allosteric inputs. Library screening experiments demonstrate that linker space is highly plastic as the induction of allosterically regulated protease switches can vary from >150-fold switch-ON to >13-fold switch-OFF solely depending on the identity of the connecting linkers and relative orientation of functional domains. In addition, Pro-rich linkers yield the most potent switches contradicting the conventional use of flexible Gly-Ser linkers. Given the ease and efficiency how functional domains can be readily recombined with any type of linker, iFLinkC is anticipated to be widely applicable to the assembly of any type of fusion protein.

中文翻译：

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iFLinkC：迭代的功能性接头克隆策略，用于接头肽与功能域的组合组装和重组。

近年来，目睹了通过蛋白质支架和零件的模块化组织工具箱的重组来工程化人工生物学功能的努力。一个关键的但经常被忽视的方面涉及连接单个结构域的肽接头或间隔子的身份，这些结构域仍然了解不多且难以组装。为解决这些局限性，iFlinkC包含高度可扩展的DNA组装过程，该过程可促进功能域与长度和灵活性不同的接头的组合重组，从而克服了GC含量高和接头元件重复性的挑战。iFLinkC的功能在构建基于PDZ-FN3的亲和钳和单链FKBP12-FRB受体作为变构输入的合成蛋白酶开关中得到了证明。文库筛选实验表明，接头空间是高度可塑性的，因为变构调节的蛋白酶开关的诱导可以从> 150倍的开通到> 13倍的关断变化，仅取决于连接接头的身份和功能的相对方向域。此外，富含Pro的接头产生最有效的开关，这与柔性Gly-Ser接头的常规用法相矛盾。鉴于功能域可以轻松地与任何类型的接头重组的简便性和效率，iFLinkC有望广泛应用于任何类型融合蛋白的组装。仅根据连接链接器的身份和功能域的相对方向进行13倍关闭。此外，富含Pro的接头产生最有效的开关，这与柔性Gly-Ser接头的常规用法相矛盾。鉴于功能域可以轻松地与任何类型的接头重组的简便性和效率，iFLinkC有望广泛应用于任何类型融合蛋白的组装。仅根据连接链接器的身份和功能域的相对方向进行13倍关闭。此外，富含Pro的接头产生最有效的开关，这与柔性Gly-Ser接头的常规用法相矛盾。鉴于功能域可以轻松地与任何类型的接头重组的简便性和效率，iFLinkC有望广泛应用于任何类型融合蛋白的组装。