High-Efficiency Transformation of Plasmid DNA into Yeast

Abstract

The yeast two-hybrid system was first introduced by Fields and Song (1) in 1989. They showed that plasmids expressing the GAL4 _BD-SNF1 and GAL4 _AD-SNF4 fusion proteins activated a GALl-lacZ reporter gene. The first application of the system involved cotransformation of two plasmids into the strain GGY1:: 171—one plasmid containing the GAL4 _BD fused to sequences from the 3′ end of SIR4 and the other containing GAL4 _AD fused to fragments of yeast genomic DNA (2). The cotransformation was accomplished by the newly reported high-efficiency lithium acetate/single-stranded carrier DNA/polyethylene glycol (LiAc/ssDNA/PEG) protocol (3) to generate 220,000 transformants and identified two positive clones. The efficiency of this transformation protocol was 2×10⁴ transformants/(g of plasmid·108 cells) (3), and the frequency of cotransformation was shown to range from 30 to 40% (4). Screening a mam-malian cDNA library by the yeast two-hybrid system requires a large number of transformants. For example, the detection of proteins that interacted with huntingtin, the Huntington disease protein, involved a screen of 4×10⁷ transformants from an adult human brain cDNA library (5). We have reported several modifications and improvements to the LiAc/ssDNA/PEG protocol so that it can easily generate the numbers of transformants required for such screens.