Construction And Expression Of "Bait Plasmid" Containing Human Secreted Form Klotho Gene In Yeast Two-hybrid System

Klotho(KL) has long been shown to be involved in aging process of animals. But the underlying mechanism is still not clearly understood.It has been identified that it is secreted form of KL that plays an important role in aging regulation.In order to elucidate the mechanism how KL works,it is necessary to find the binding protein of KL.So we decided to construct a bait vector containing human secreted form klotho gene(hsKL) in yeast two-hybrid system to screen the cDNA library of Human. The self-activation and toxicity of the bait protein in yeast cell was analyzed.The human secreted form klotho gene fragment(hsKL) was amplified from the pcDNA3.1-KL by PCR.The product was inserted into pMD18-T vector.After conforming by sequencing,the target fragent was inserted into the"bait plasmid"pGBKT7(named as pGBKT7-kl).After confirmation with restricted endonuclease digestion and sequence analysis,the plasmid was transformed into the yeast cell AH109,and its toxicity and transcriptional activation was tested by the phenotype assay.The yeast cell AH109 transfected with pGBKT7-hsKL couldn't grow on the SD/-His/-Trp/-Leu medium,which suggested that the expression of the bait plasmid can not activate the transcription of report gene alone.Besides,the transfection has no toxicity to the yeast cell.The bait plasmid pGBKT7-kl constructed expresses correctly,and has no self-activation and toxicity in yeast two-hybrid system.So it can be used for downstream screening hsKL-binding proteins.