We report the development of soluble expression phage-assisted continuous evolution (SE-PACE), a system for rapidly evolving proteins with increased soluble expression. Through use of a PACE-compatible AND gate that uses a split-intein pIII, SE-PACE enables two simultaneous positive selections to evolve proteins with improved expression while maintaining their desired activities. In as little as three days, SE-PACE evolved several antibody fragments with >5-fold improvement in expression yield while retaining binding activity. We also developed an activity-independent form of SE-PACE to correct folding-defective variants of maltose-binding protein (MBP) and to evolve variants of the eukaryotic cytidine deaminase APOBEC1 with improved expression properties. These evolved APOBEC1 variants were found to improve the expression and apparent activity of Cas9-derived base editors when used in place of the wild-type cytidine deaminase. Together, these results suggest that SE-PACE can be applied to a wide variety of proteins to rapidly improve their soluble expression.

我们报告了可溶性表达噬菌体辅助连续进化（SE-PACE）的发展，这是一种快速进化蛋白质并增加可溶性表达的系统。通过使用与 PACE 兼容的 AND 门（使用分裂内含肽 pIII），SE-PACE 能够同时进行两个正向选择，以进化出表达改善的蛋白质，同时保持其所需的活性。在短短三天内，SE-PACE 进化出了多个抗体片段，表达产量提高了 5 倍以上，同时保留了结合活性。我们还开发了一种活性独立形式的 SE-PACE，以纠正麦芽糖结合蛋白 (MBP) 的折叠缺陷变体，并进化出具有改进表达特性的真核胞苷脱氨酶 APOBEC1 变体。这些进化的 APOBEC1 变体被发现在代替野生型胞苷脱氨酶时可以提高 Cas9 衍生碱基编辑器的表达和表观活性。总之，这些结果表明 SE-PACE 可应用于多种蛋白质，以快速提高其可溶性表达。