Screening of candidate proteins interacting with lung cancer metastasis-related protein 1 from human liver cDNA library by yeast two hybridization

Objective To investigate the biological function of the lung cancer metastasis-related protein 1(LCMR1) by screening proteins interacting with LCMR1 from human liver cDNA library performed by yeast two-hybrid system. Methods The coding sequences of LCMR1 was cloned into the pGBKT7 and the bait plasmid pGBKT7-LCMR1 was constructed. Then the bait plasmid was transformed into AH109 yeast strains, and expression of LCMR1 fusion protein, transcriptional activation and toxicity were tested. A yeast two-hybrid screening was performed by mating AH109 with Y187 containing human liver cDNA library plasmids. After eliminating false positive clones, the interaction between LCMR1 and proteins obtained from positive colonies were further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from positive colonies, we carried out a bioinformatic analysis. Results The bait plasmid pGBKT7-LCMR1 was successfully constructed and transformed into yeast strain AH109, and this recombinant plasmid pGBKT7-LCMR1 showed no self-activation and toxicity in AH109 yeast. After repeating yeast two-hybrid screen, 14 true positive colonies were selected and sequenced. Among the 14 positive colonies, 7 coding genes with known functions were obtained through sequencing and bioinformatics analysis, which were RPL29, RPS15a, HAMP, Complement C3, ND4L, ND1 and CYP2E1. Conclusion Genes of proteins interacting with LCMR1 were successfully screened out from human liver cDNA library by yeast two-hybrid system.