The discovery of the interaction between augmenter of liver regeneration and Na+, K+-ATPase with yeast two-hybrid system

AIM:To screen the proteins interacting with human augmenter of liver regeneration (hALR) by yeast two-hybrid system and to study the mechanism of hALR action. METHODS:hALR bait plasmid was constructed by ligating the gene of hALR into pGBKT7, then transformed into yeast AH109. The yeast strain AH109 containing pGBKT7-hALR was mated with yeast Y187 containing human liver cDNA library plasmid. Diploid yeast was plated on SD/-trp-leu-his-ade (QDO) for screening and on QDO containing X-α-gal for further selection.The AD/library inserts were amplified by PCR and the PCR products were characterized by digesting with Sau3AⅠ and HaeⅢ restriction enzyme to eliminate the duplicates. After sequencing, the positive clones were analysed by bioinformatics. RESULTS:Several positive clones were obtaind. The sequencing and analysis shown that one of them is 669 bp DNA fragment encoding β subunit of Na⁺, K⁺-ATPase. The 224 bp 3'terminal DNA fragment is non-encoder region, and the 445 bp 5'terminal DNA encodes C-terminal 147 amino acid residues of Na⁺, K⁺-ATPase β subunit. CONCLUSION:The results of screening proteins using yeast two-hybrid system showed that hALR could interact directly with Na⁺, K⁺-ATPase in the yeast cell.