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 polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 × 106 combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.

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Figure 1: (A) DNA constructs code for fusions between library proteins (shown as α-helical leucine zippers) and either fragment of murine DHFR (mDHFR).
Figure 2: (A) Schematic representation of a leucine zipper pair visualized from the N-terminus illustrating e/g-interactions and the hydrophobic core formed by the a- and d-positions.
Figure 3: Efficiency of competition in a model selection.
Figure 4: Competition selection and chain shuffling.
Figure 5: Sequencing profile of pools from passages of the chain shuffling WinZip-B1-DHFR[2:I114A] + LibA-DHFR[1].


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J.N.P. was a recipient of a fellowship from les Fonds de la Recherche en Santé du Québec, and currently holds a fellowship from le Conseil de Recherche en Sciences Naturelles et en Génie du Canada. K.M.A. is a recipient of a doctoral grant from the Stipendienfonds der Basler Chemischen Industrie. This work was funded by The Burroughs-Wellcome Fund (S.W.M.) and by grant 0311628 from the Bundesministerium für Bildung und Forschung (AP). We thank Günter Wellnhofer (Morphosys AG, Munich, Germany) for the synthesis of the oligonucleotides with the mixed trinucleotide building blocks. We thank Michel Denault for his painstaking help in probabilistic analysis, and François-Xavier Campbell-Valois, Alexis Vallée-Belisle, and Patrick Forrer for critical reading of the manuscript.

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Correspondence to Stephen W. Michnick.

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