bHLHZ/bZIP Hybrid proteins evolved to target a specific DNA site in a modified-yeast one-hybrid system

ORGN 634

Jing Xu, jxu24@utm.utoronto.ca, Hiu Kwan Chow, and Jumi A. Shin, jshin@utm.utoronto.ca. Department of Chemistry, University of Toronto, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada
We developed a modified yeast one-hybrid (Y1H) system that enables us to perform in vivo directed evolution of proteins targeting a desired DNA site. We started with the minimalist protein Max-C/EBP, a hybrid of the basic region from the bHLHZ protein Max and leucine zipper from bZIP protein C/EBP. Max-C/EBP did not bind E-box (5'-CACGTG) in the Y1H assay. The E-box is targeted by the proto-oncogenic Myc, Max, Mad network involved in diverse cellular functions. After rounds of selection, we uncovered mutants with significant binding capability. Our system is particularly useful for improvement of the function of a rationally designed protein. Details of evolved proteins will be presented.