The creation of novel protein functions represents a significant challenge in protein engineering. To address this challenge, we developed a simple and efficient method – in vitro coevolution – for the creation of novel protein functions in an existing protein scaffold. The in vitro coevolution method involves the design of a hypothetical pathway toward the target protein function followed by stepwise directed evolution of an appropriate protein scaffold along the pathway. As proof of concept, we applied this strategy to the engineering of two proteins. In the first example, in vitro coevolution was used to engineer variants of the human estrogen receptor alpha ligand binding domain (hERaLBD) with novel corticosterone activity. Two steroidal ligands – testosterone and progesterone – that provide a progressive structural bridge between the natural ligand 17b-estradiol and the target ligand corticosterone, were chosen to assist the directed evolution of hERaLBD. A total of approximately 10^6 randomly point-mutated variants were phenotypically screened in four rounds of directed evolution, resulting in two hERaLBD variants that respond to corticosterone. Creation of this new ligand activity required the presence of four simultaneous mutations, whereas individual mutations had no effect. In addition, several required mutations were located outside the ligand binding pocket and yet exerted important action on ligand binding. Our results demonstrate the ability of in vitro coevolution to create novel protein function that is difficult or impossible to achieve by existing protein engineering approaches. In the second example, in vitro coevolution was used to the engineer variants of homing endonuclease I-SceI with novel DNA cleavage activity. Homing endonucleases have great potential for applications such as targeted gene correction in gene therapy and gene alteration in systems biology and metabolic engineering. However, the limited repertoire of recognition sequences in naturally available homing endonucleases severely hampers their usefulness. We developed a highly sensitive selection method for the directed evolution of homing endonucleases that couples enzymatic DNA cleavage with the survival of host cells. Using this selection method and in vitro evolution, we attempted to engineer I-SceI variants that can selectively cleave a target sequence found in a mutant transmembrane conductance regulator (CFTR) gene involved in cystic fibrosis. So far, we have successfully completed two rounds of directed evolution toward the first intermediate target sequence, and obtained a variant that has ~5000 fold increased activity toward the new target sequence compared to the wild type enzyme.

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