Yeast Two-Hybrid Screening

To perform the yeast two-hybrid screening, IpaH4.5 was inserted into the bait pGBKT7 vector for expression as a fusion protein with the Gal4 DNA binding domain (Gal4-BD). This plasmid was used to transform the AH109 yeast strain, and Gal4-BD fused IpaH4.5 was used as bait for the screening of human spleen cDNA library. The spleen cDNA library was inserted into the pGADT7 vector (Clontech) for expression as fusions with the Gal4 activation domain (Gal4-AD) and was maintained in the Y187 strain of yeast. Transformed AH109 and Y187 yeast cells were mixed together for mating. Positive clones were selected on synthetic dropout medium lacking 4 nutrients (Leu/Trp/Ade/His). The blue colonies were kept, and the positive results were confirmed by repeating assays. cDNA plasmids isolated from positive colonies were introduced into Escherichia coli DH5α and sequenced. The sequences were analyzed with the BLAST program in NCBI.