Evolution of BoNT peptidases

A method for evolving a Botulinum neurotoxin (BoNT) protease, the method comprising:

• (a) contacting a population of host cells with a population of phage vectors comprising a gene encoding a BoNT B, BoNT D, BONT E, or BoNT F protease and being 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 BoNT 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 BoNT protease, and the level of gene product expression depends on the activity of the BoNT protease;
• (b) incubating the population of host cells under conditions allowing for mutation of the gene encoding the BoNT protease and the transfer of the vector comprising the gene encoding the BONT 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 an evolved BONT B, BoNT D, BONT E, or BoNT F protease,
• wherein the host cell expresses a fusion protein comprising:
• (i) a transcriptional activator;
• and
• (ii) an inhibitor of the transcriptional activator, wherein the inhibitor is fused to the transcriptional activator via a linker comprising a BoNT protease cleavage site that is cleaved by the evolved BONT protease;
• and
• wherein the BoNT protease cleavage site comprises the sequence:
• NGSLCDQEIDSICSIERADN (SEQ ID NO:
• 311) or GGSGGSGGSKGLDKVMETQAQVDELKGIMVRNIDLVAQRGERLELLIDKTENLVDSSVTF KTTSRNLARGGSGGSGGS (SEQ ID NO:
• 313).