Screening Of Candidate Proteins Interacting With Lung Cancer Metastasis-related Protein 1 (LCMR1)

Objective: The gene of LCMR1 (lung cancer metastasis-related protein 1) is a novel one that was cloned with a differential display polymerase chain reaction (DD-PCR) technique in our laboratory (GenBank accession: AY 148462). In order to investigate the biological function of the LCMR1, we screened proteins interacting with LCMR1 from human liver cDNA library by yeast two-hybrid system.Methods: The LCMR1 gene was amplified by PCR from the pGEX-5T-LCMR1 vector constructed before, and the EcoRI and PstI sites were introduced into it. The LCMR1 gene, after sequenced, was cloned into pGBKT7 (BD), then "bait" plasmid pGBKT7- LCMR1 was transformed into the yeast strain AH109. Following that, the toxicity and self-activation of this recombinant plasmid pGBKT7-LCMR1 in AH109 yeast were tested. After expression of the pGBKT7-LCMR1 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH109 with Y187 containing human liver cDNA library plasmids (AD). The mated yeast was plated on quadruple dropout medium and assayed for α-galactosidase activity. The interaction between LCMR1 and proteins obtained from positive colonies was 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: (1) The "bait" plasmid pGBKT7-LCMR1 was successfully constructed and transformed into yeast strain AH109. Also, this recombinant plasmid pGBKT7- LCMR1 had no self-activation and toxicity in AH109 yeast. (2) After our repeating the yeast two-hybrid screen, 14 true positive colonies were selected and sequenced. Among the 14 positive colonies, 7 coding genes withknown functions were obtained through sequencing and bioinformatics analysis, which were RPL29. RPS15a, HAMP, Complement C3, ND4L, ND1 and Cytochrom P450.Conclusions: (1) The "bait" plasmid pGBKT7-LCMR1 without self-activation and toxicity in AH109 yeast was successfully constructed; (2) Genes of proteins interacting with LCMR1 were successfully screened from human liver cDNA library, they are RPL29, RPS15a, HAMP, Complement C3, ND4L, ND1 and CYP450.