Construction of a bait plasmid of human LRP16 gene in yeast two-hybrid system

Objective: To construct a bait plasmid of LRP16 gene in yeast cells for probing the proteins that could interact with LRP16 by screening in human cDNA library.Methods: The LRP16 fragment that could encode full-length protein sequence was recovered by restriction digestion from pLPC-LRP16 plasmid,and subsequent electrophoresis and reclamation.The fragment was inserted into bait vector pGBKT7 of yeast two-hybrid system at the EcoR I sites.Following confirming the correct insertion direction by the appropriate restriction enzymes,the recombined construct was transformed into yeast cells AH109 and Y187,respectively.The toxicity and the self-activating transcriptional activation of human LRP16 protein in these two yeast cells were firstly detected.The ectopic expression of human LRP16 protein in the total yeast protein extracts was also measured by western blot analysis.Results: The insertion direction and size of human LRP16 in the recombined plasmid were both correct,the toxicity and self-activating transcriptional activation of human LRP16 protein were not observed in yeast cells.Importantly,the human LRP16 protein could be effectively expressed in transformed yeast.Conclusions: The construct of pGBKT7-LRP16 can correctly encode human LRP16 protein in yeast cells,and can be used as a bait in yeast two-hybrid system.