Evolution of proteases

A method of generating protease variants that are resistant to a protease inhibitor, the method comprising:

• (a) contacting a population of host cells with a population of phage vectors comprising a gene encoding a protease and deficient in at least one gene for the generation of infectious phage particles, wherein:
• (1) the host cells are amenable to transfer of the vector;
• (2) the vector allows for expression of the protease in the host cell and can be replicated by the host cell, and the replicated vector can transfer into a second host cell;
• and
• (3) the host cell expresses a gene product encoded by the at least one gene for the generation of infectious phage particles of (a) in response to the activity of the protease, and the level of gene product expression depends on the activity of the protease;
• wherein the host cell expresses a fusion molecule comprising:
• (i) a transcriptional activator, wherein the transcriptional activator is an RNA polymerase, and
• (ii) an inhibitor of the transcriptional activator of (i), wherein the inhibitor is fused to the transcriptional activator of (i) via a linker comprising a protease cleavage site that is cleaved by the protease,
• (b) incubating the population of host cells in the presence of a protease inhibitor and under conditions allowing for mutation of the gene encoding the protease and the transfer of the vector comprising the gene encoding the protease from host cell to host cell, wherein host cells are removed from the host cell population, and the population of host cells is replenished with fresh host cells that do not harbor the vector;
• and
• (c) isolating a replicated vector from the host cell population in (b), wherein the replicated vector comprises a mutated version of the gene encoding a protease variant, wherein the protease variant exhibits increased protease activity in the presence of the protease inhibitor as compared to the version of the protease from (a).