Screening of FOXP3-interacted proteins by yeast two-hybrid technique⋆

ObjectiveTo screen the proteins interacting with the Treg specification factor forkhead box protein P3 (FOXP3) by yeast two-hybrid system.MethodsHuman FOXP3 gene was amplified by nest RT-PCR from peripheral blood mononuclear cells (PBMC) and inserted into plasmid pGBKT7 to construct the bait vector, then the self-activation and toxicity of the bait vector in host yeast strain AH109 were observed. Thereafter, a human liver cDNA library was screened by the bait vector. The positive clones were selected out by nutrient-deficient culture and back-hybridizing. The sequences from the candidate positive clones were blasted and analyzed by bioinformatics methods.ResultsThe constructed bait vector encoding FOXP3 was found no self-activation and toxicity in yeast AH109. Three proteins which interacted with FOXP3, including tumor protein D52, splicing factor 3b subunit 1 and hypothetical protein, were identified.ConclusionThree new candidate proteins interacting with FOXP3 are selected out by this yeast two-hybrid system and library, which may facilitate the further study of FOXP3 in Treg.