Yeast two-hybrid screening

Description

The yeast two-hybrid system is a powerful tool for rapid screening of protein-protein interactions. Yeast two-hybrid libraries constructed from Penaeus monodon postlarvae, lymphoid organ and ovary were established in our laboratory. WSSV ORFs were also cloned into the GAL4 DNA binding domain vector (pGBKT7) and GAL4 activation domain vector (pGADT7). These libraries can be used to identify WSSV proteins that interest with shrimp proteins or with other WSSV proteins. So far, we have successfully used the yeast two-hybrid technique to analyze the interaction between WSSVs structured proteins.

Brief protocol

The bait vector BD-X was used to screen independent recombinant clones of the shrimp cDNA library in Saccaromyces cerevisiae strain Y187. After mating the library host strain with the bait strain, positive clones were selected for growth on a synthetic dropout (SD) plate lacking Adenine, Histidine, Leucine and Tryptophan (SD/-Ade/-His/-Leu/-Trp) and containing X-α-Gal. Plasmid DNA was isolated from those clones that activated all four yeast reporter genes (ADE2; HIS3; MEL1, encoding α-galactosidase; and lacZ, encoding β-galactosidase) and transformed into E. coli strain DH5α to recover the plasmid for sequencing. To confirm the screening result, recovered plasmids (containing both BD-X and AD-Y) were re-transformed back into a host strain and plated on SD/-Ade/-His/-Leu/-Trp. Positive interactions were re-tested by cotransforming bait and prey plasmids into Saccaromyces cerevisiae strain AH109 according to the manufacturer’s instructions. For the positive control, the AH109 cells were cotransformed with plasmids pGBK-53 (p53) and pGAD-RecT (simian virus 40 large T antigen). Negative controls employed pGBKT7 (BD only) and pGADT7-Rec (AD only).

Some of our publications that have used this platform

1. Chang YS, Liu WJ, Lee CC, Chou TL, Lee YT, Wu TS, Huang JY, Huang WT, Lee TL, Kou GH, Wang AH, Lo CF. (2010) A 3D model of the membrane protein complex formed by the white spot syndrome virus structural proteins. PLoS One 5: e10718.
2. Chang YS, Liu WJ, Chou TL, Lee YT, Lee TL, Huang WT, Kou GH, Lo CF. (2008) Characterization of white spot syndrome virus envelope protein VP51A and its interaction with viral tegument protein VP26. J Virol 82: 12555-12564.
3. Chen LL, Lu LC, Wu WJ, Lo CF, Huang WP. (2007) White spot syndrome virus envelope protein VP53A interacts with Penaeus monodon chitin-binding protein (PmCBP). Dis Aquat Organ 74: 171-178.