Inteins are self-splicing proteins that exist as internal insertion sequences in host proteins. In general, intein insertion prevents the function of the host protein when the fusion protein is translated, but subsequent splicing restores the host protein function. Thus, inteins have the potential to be used as post-translational molecular "switches" for the control and delivery of various target proteins. Laboratory evolution is widely used to generate proteins with improved or modified functions. To apply this strategy to engineer novel inteins functioning under desirable environments or responding to specific molecules, we have developed a genetic selection system using phage display for the selection of enhanced splicing activity under a wide range of conditions. First, as a control experiment, a series of mini-inteins was inserted into a chitin-binding domain (ChBD), and displayed on the surface of T7 phage. The phages displaying ChBD disrupted by an inactive intein had low affinity for chitin, while the phages displaying an active intein had high affinity, since ChBD was reconstructed by splicing. The system was robust under in vivo and various in vitro selection conditions. Second, a randomly mutated mini-intein library was displayed as a fusion with ChBD. After several rounds of selection, we successfully isolated intein mutants with significantly enhanced splicing activity, over a broad range of conditions. Some of the mutants exhibited higher activity than the most active mini-intein we had previously isolated using an alternate in vivo selection system. The key mutations have been mapped on the previously determined intein structure. The molecular evolution strategy described here is applicable to the creation of novel intein-based molecular tools, such as molecular sensors or regulators triggered by a number of different chemical or physical signals.

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