Transcriptional activation function of intracellular domain of EPOR and G-CSFR protein in yeast: a comparative study / 解放军医学杂志

ABSTRACT

Objective To investigate the feasibility of searching for proteins which interact with intracellular domain of hematopoietic growth factor receptors using yeast two-hybrid system. Methods RT-PCR method was performed to amplify the genes of intracellular domains of G-CSF receptor and EPO receptor in NFS-60 and BET-2 cells of mice. The genes were cloned into yeast expression plasmid pGBKT7 vector,and then transformed into yeast AH109. The yeast proteins were isolated and analyzed with Western blotting. Transcriptional activation was analyzed by the ?-galactosidase colony-lift filter assay. Results The intracellular domains of G-CSF receptor and EPO receptor genes were successfully cloned into pGBKT7 vector. The results of Western blotting assay showed that both proteins were expressed in the yeast cells. The ?-galactosidase colony-lift filter assay demonstrated that G-CSF receptor alone had no activity of transcriptional activation,while the EPO receptor alone could activate transcription. Conclusion The findings suggested that intracellular domain of G-CSF receptor could be used as a bait to find interacting proteins using yeast two-hybrid system,while that of the EPO receptor could not. Therefore the system could not be applied to all hematopoietic factor receptor.