Directed evolution is widely used to optimize protein folding and solubility in cells. Although the screening and selection of desired mutants is an essential step in directed evolution, it generally requires laborious optimization and/or specialized equipment. With a view toward designing a more practical procedure, we previously developed an inducible plasmid display system, in which the intein (auto-processing) and Oct-1 DNA-binding (DBD) domains were used as the protein trans-splicing domain and DNA-binding module, respectively. Specifically, the N-terminal (CfaN) and C-terminal (CfaC) domains of intein were fused to the C-terminal end of the His-tag and the N-terminal end of Oct-1 DBD to generate His₆-CfaN and CfaC-Oct-1, respectively. For such a system to be viable, the efficiency of protein trans-splicing without the protein of interest (POI) should be maximized, such that the probability of occurrence is solely dependent on the solubility of the POI. To this end, we initially prevented the degradation of l-arabinose (the inducer of the P_BAD promoter) by employing an Escherichia coli host strain deficient in the metabolism of l-arabinose. Given that a low expression of His₆-CfaN, compared with that of CfaC-Oct-1, was found to be conducive to the generation to a soluble product of the protein trans-splicing event, we designed the expression of His₆-CfaN and CfaC-Oct-1 to be inducible from the P_BAD and P_T7 promoters, respectively. The optimized system thus obtained enabled in vitro selection of the plasmid–protein complex with high yield. We believe that the inducible plasmid display system developed in this study would be applicable to high-throughput screening and/or selection of protein variants with enhanced solubility.

定向进化广泛用于优化细胞中的蛋白质折叠和溶解度。虽然筛选和选择所需的突变体是定向进化的必要步骤，但它通常需要费力的优化和/或专门的设备。为了设计更实用的程序，我们之前开发了一种诱导型质粒展示系统，其中使用内含肽（自动加工）和 Oct-1 DNA 结合（DBD）域作为蛋白质反式剪接域和 DNA -binding 模块，分别。具体而言，intein 的 N 端 (CfaN) 和 C 端 (CfaC) 结构域与 His 标签的 C 端和 Oct-1 DBD 的 N 端融合以生成 His ₆-CfaN 和 CfaC-Oct-1，分别。为了使这样的系统可行，没有感兴趣的蛋白质（POI）的蛋白质反式剪接的效率应该最大化，使得发生的概率仅取决于POI的溶解度。为此，我们最初通过使用缺乏l-阿拉伯糖代谢的大肠杆菌宿主菌株来防止l-阿拉伯糖（P _BAD启动子的诱导剂）的降解。鉴于与 CfaC-Oct-1 相比，His ₆ -CfaN的低表达有助于产生蛋白质反式的可溶性产物-剪接事件，我们设计了 His ₆ -CfaN 和 CfaC-Oct-1 的表达，分别由P _BAD和P _T7启动子诱导。由此获得的优化系统能够以高产率在体外选择质粒-蛋白质复合物。我们相信本研究中开发的诱导型质粒展示系统将适用于高通量筛选和/或选择具有增强溶解度的蛋白质变体。