(13a) Protein Switches Created by Non-Homologous Recombination

We have developed a directed
evolution approach for creating protein switches involving the in vitro recombination of two non-homologous genes with the
prerequisite input and output functions. We have recombined the genes coding
for TEM1 b-lactamase (BLA) and the E. coli maltose binding protein (MBP) to create a family of MBP-BLA
hybrids in which maltose is either a positive or negative effector of b-lactam
hydrolysis. Some of these MBP-BLA switches were effectively 'on-off' in
nature, with maltose altering catalytic activity by as much as 600-fold. One
MBP-BLA switch was identified that could be positively allosterically regulated
by maltose and negatively allosterically regulated by Zn²⁺, which is
surprising considering that neither BLA nor MBP bind Zn²⁺. The
origin of this effect has implications for how novel protein function evolves.
The ability of the MBP-BLA switches to confer an effector-dependent growth/no
growth phenotype to E. coli cells was
exploited to rapidly identify, from a library of 4 x 10⁶ variants,
MBP-BLA switch variants that respond to sucrose as the effector. The
transplantation of these mutations into wildtype MBP converted MBP into a
'sucrose-binding protein,' illustrating the switches potential as a tool to
rapidly identify novel ligand-binding proteins.