Vivo library-versus-library selection of optimized protein-protein interactions
US6897017B1 · kind B1 · utility
Assignee
Inventors
Key dates
| Filing date | Jun 26, 2000 |
| Grant date | May 24, 2005 |
| Priority date | — |
| Expiry date | Jun 26, 2020 |
Classification
- Technology area (CPC Y)Emerging Cross-Sectional Technologies
- CPC primaryY02A90/10
- WIPO fieldMeasurement
- WIPO sectorInstruments
Abstract
The present invention describes a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. It allows for the screening of a protein library against a second protein library, rather than against a single bait protein, and thus has numerous applications in the study of protein-protein interactions. Additionally, it allows for the application of different selection stringencies. 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 E. coli. Interaction between the library polypeptides was required for reconstitution of the 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 multip…
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