Dodevski Igor, Markou George C, Sarkar Casim A
Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455, USA.
Curr Opin Struct Biol. 2015 Aug;33:1-7. doi: 10.1016/j.sbi.2015.04.008. Epub 2015 Jun 18.
Although cell-free directed evolution methods have been used to engineer proteins for nearly two decades, selections on more complex phenotypes have largely remained in the domain of cell-based engineering approaches. Here, we review recent conceptual advances that now enable in vitro display of multimeric proteins, integral membrane proteins, and proteins with an expanded amino acid repertoire. Additionally, we discuss methodological improvements that have enhanced the accessibility, efficiency, and robustness of cell-free approaches. Coupling these advances with the in vitro advantages of creating exceptionally large libraries and precisely controlling all experimental conditions, cell-free directed evolution is poised to contribute significantly to our understanding and engineering of more complex protein phenotypes.
尽管无细胞定向进化方法已被用于蛋白质工程近二十年，但对更复杂表型的筛选在很大程度上仍局限于基于细胞的工程方法领域。在此，我们回顾了近期的概念性进展，这些进展现在使得多聚体蛋白、整合膜蛋白以及具有扩展氨基酸库的蛋白能够在体外展示。此外，我们还讨论了方法学上的改进，这些改进提高了无细胞方法的可及性、效率和稳健性。将这些进展与创建超大文库以及精确控制所有实验条件的体外优势相结合，无细胞定向进化有望为我们对更复杂蛋白质表型的理解和工程化做出重大贡献。
