In vivo library-versus-library selection of optimized protein-protein interactions

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 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 2.0×10 6 combinations, and selected a novel leucine zipper pair which may be appropriate for use in further in vivo heterodimerization strategies.

1 . A method for identifying an interacting set of molecules comprising:
 A) generating fragments of a reporter molecule which have a directly or indirectly detectable activity when associated;   B) coupling first fragments to members of a first panel of molecules;   C) coupling second fragments to members of a second panel of molecules;   D) mixing the products of B) and C);   E) directly or indirectly testing for said activity; and   F) identifying the panel members whose interaction resulted in said activity and which thus form an interacting set.   
   
   
       2 . A method for identifying an interacting set of molecules comprising:
 A) identifying a first and a second panel of molecules whose mutual interaction is desired to be tested;   B) coupling molecules of said first panel to first fragments of a protein reporter molecule;   C) coupling molecules of said second panel to second fragments of said protein reporter molecule;   D) mixing the products of B) and C);   E) directly or indirectly testing for said activity; and   F) identifying the panel members whose interaction resulted in said activity and which thus form an interacting set.   
   
   
       3 . A method of  claim 1  where at least one of said panels comprises a library of molecules. 
   
   
       4 . A method of  claim 1  where at least two of said panels comprise a library of molecules. 
   
   
       5 . A method of  claim 2  where at least one of said panels comprises a library of molecules. 
   
   
       6 . A method of  claim 2  where at least two of said panels comprise a library of molecules. 
   
   
       7 . A method of screening multiple panels of molecules against each other to determine the ability of individual panel members to form an interacting set comprising:
 A) coupling first and second fragments of a protein reporter molecule to different panel members;   B) mixing the products of A);   C) testing for reporter molecule activity; and   D) identifying the panel members whose interaction results in said activity and which thus form an interacting set.   
   
   
       8 - 17 . (canceled)