Screening The Interacting Proteins Of Myosin X By Yeast Two-hybrid System

Yeast two-hybrid system is one of the most important methods to study protein-protein interaction. The system is sensitive, simple and rapid, which the sequence of target proteins can be obtained directly. For the last few years, yeast two-hybrid system is used to study protein-protein interaction, screen the novel proteins and study the function of protein.Myosin X is one of the members in unconventional myosin super-family and expresses at low concentrations in most vertebrate, which is relative to filopodial formation, neuronal growth, cell migration, cell adhesion and the spindle formation of mitosis. In this study, yeast two-hybrid technique was used to identify proteins interacting with Myo X. Here, the MyTH4 domain and FERM domain of Myo X tail are a bait to screen the human fetal brain cDNA library. Plating the cells on SD without histidine, tryptophan, leucine and adenine to assay the co-transformants to express the report genes and detect the expression of lac Z gene, a total of 112 positive clones were obtained, then, the positive clones were cultured in SD medium without histidine, tryptophan,leucine and adenine five times to remove the false clones. The backcross experiment was employed to ensure the results. The plasmid was extracted from yeast, and then it was transformed into E. coli and the target plasmid was obtained by selective culture. The cDNA segment of the positive clones were sequenced and compared with GENE BANK. The proteins screened in this study were probably involved in cell migration, cell adhesion, cystoskeleton rearrangement, protein transportation, regulation of gene expression and signal transduction.