Yeast Two-Hybrid Screening for Tat-Binding Proteins

Yeast two-hybrid screening was conducted using the Matchmaker GAL4 two-hybrid system 3 (Clontech) as described previously [54 (link)]. In brief, the region containing full-length Tat was generated by PCR, cloned downstream of the GAL4 DNA-binding domain in pGBKT7 (pGBKT7–Tat/bait), and introduced into the yeast strain PBN204 containing three reporters (URA3, ADE2, and LacZ), which are under the control of different GAL promoters. The transformants of the Tat bait and the human thymus cDNA activation domain library were screened on selection medium lacking leucine, tryptophan, and uracil, which support the growth of yeast when the bait plasmid and the prey proteins interact with each other. After selection of the putative Tat-binding protein, NUCKS1, the amplified NUCKS1-expressing prey plasmid was reintroduced into the yeast PBN204 strain with the Tat bait plasmid to confirm the interaction of Tat–NUCKS1.