Department of Life Technologies, University of Turku Kiinamyllynkatu 10, 20520 Turku, Finland.
ACS Synth Biol. 2022 Oct 21;11(10):3174-3181. doi: 10.1021/acssynbio.2c00260. Epub 2022 Sep 30.
DsbA leader peptide targets proteins for cotranslational translocation by signal recognition particle (SRP) pathway and has been the standard signal sequence for filamentous phage display of fast-folding Designed Ankyrin Repeat Proteins (DARPins). In contrast, translocation of DARPins via the post-translational pathway, for example, with the commonly used PelB leader, has been reported to be highly inefficient. In this study, two PelB signal sequence libraries were screened covering different regions of the leader peptide for identifying mutants with improved display of DARPins on phage. A PelB variant with the most favorable combination of synonymous mutations in the n-region and hydrophobic substitutions in the h-region increased the display efficiency of a DARPin library 44- and 12-fold compared to PelB and DsbA, respectively. Based on thioredoxin-1 (TrxA) export studies the triple valine mutant PelB DN5 V3 leader was capable of more efficient cotranslational translocation than PelB, but the overall display efficiency improvement over DsbA suggests that besides increased cotranslational translocation other factors contribute to the observed enhancement in DARPin display efficiency.
DsbA 信号肽可将蛋白质靶向用于信号识别颗粒（SRP）途径的共翻译易位，并且一直是用于快速折叠的设计锚蛋白重复蛋白（DARPins）的丝状噬菌体展示的标准信号序列。相比之下，通过翻译后途径（例如，使用常用的 PelB 信号肽）易位 DARPins 的效率非常低。在这项研究中，筛选了两个 PelB 信号序列文库，涵盖了该信号肽的不同区域，以鉴定提高噬菌体上 DARPins 展示效率的突变体。在 n 区具有最有利的同义突变组合且在 h 区具有疏水性取代的 PelB 变体与 PelB 和 DsbA 相比，分别将 DARPin 文库的展示效率提高了 44 倍和 12 倍。基于硫氧还蛋白-1（TrxA）出口研究，三缬氨酸突变体 PelB DN5 V3 信号肽能够比 PelB 更有效地共翻译易位，但相对于 DsbA 的整体展示效率提高表明，除了增加共翻译易位外，其他因素也有助于观察到 DARPin 展示效率的提高。
