Screening and preliminary analysis of PRR11 interacting proteins based on the yeast two-hybrid technique

Abstract:

Objective：To screen PRR11 interacting proteins by the yeast two-hybrid technique，and to provide a basis for further stud－ies of its molecular mechanism of action. Methods：PRR11 bait plasmids were made for the yeast two-hybrid system and tested for auto-activation. Mutants were constructed to obtain lower auto-activation. The human fetal brain cDNA library was screened to obtain initial positive clones. The lacZ reporter gene assay，retransformation，positive clone sequencing，and BLAST sequence alignment analysis were used to rule out false positive and repeated clones and identify candidate PRR11 interacting proteins. Results：PRR11 showed auto-activation and a PRR11 mutant with lower auto-activation was constructed. The yeast two-hybrid screening resulted in 102 initial positive clones. The lacZ reporter gene assay further validated 39 positive clones. According to gene sequencing and se－quence alignment analysis，the 39 clones were from genes encoding 15 different proteins. Finally，four candidate proteins including RNF41 were verified by retransformation. Conclusion：RNF41，SCG5，BCYRN1，and PRR13 are potential PRR11 interacting proteins.