An in vivo library-versus-library selection of optimized protein-protein interactions

We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing po lypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of t wo designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library p olypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial g rowth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent w ith selection for stable, heterodimerizing pairs. Using more weakly associa ting mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pool ed, selected colonies in liquid culture, as the best pairs allowed for bett er bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enr iched at different rates. We applied these selection processes to a library -versus-library sample of 2.0 x 10(6) combinations and selected a novel leu cine zipper pair that may be appropriate for use in further in vivo heterod imerization strategies.