| 초록 |
In this study, using a soluble methane monooxygenase (sMMO) as a model, we propose an integrative strategy for cell-free engineering of enzymes. The nucleotides of the targeted sites of sMMO were randomized and cloned into E. coli cells for the separation of individual variant genes. After being amplified by colony-PCR, the variant genes were then directly expressed in a cell-free synthesis system prepared in microtiter plates. When combined with a plate-based colorimetric screening scheme, cell-free expression and analysis of individual variant genes were completed in a matter of hours. Our results demonstrate that the techniques of cell-free protein synthesis can be effectively implemented for rapid materialization and analysis of diversified genetic information, which provides a route to optimizing enzymes for various applications. |
