Screening Of Candidate Molecules Interact With Protein Kinase Wee1B From Human Ovarian CDNA Library And The Validation Of Interaction

ObjectiveScreening of new candidate molecules interact with protein kinase Wee1Band confirming the interaction and mechanism of the target protein and Wee1B,and to further explore how it affects the early development of mouse embryos.MethodspGBKT7-Wee1B bait plasmid was constructed and transfected into yeastcells to detect its toxicity, activation and expression in yeast. Positive cloneswere extracted using yeast two hybrid system and transfected them into E.coli,and sequenced for identification. Finally, the interaction between the candidatemolecules and the Wee1B protein was verified by immunoprecipitation andimmunofluorescence in mammalian cells.Results1. pGBKT7-Wee1B bait plasmid was constructed using pcDNA3.1/V5-His-TOPO-Wee1B-WT plasmid as templates.2. pGBKT7-Wee1B plasmid is no toxic and no activation to yeast cell.Furthermore, Wee1B is expressed in yeast cells.3. Nine interacting proteins were screened from human ovarian cDNAlibrary using yeast two hybrid system.4. Wee1B was interacted with KH domain containing, RNA binding, signal transduction-associated protein3(KHDRBS3) in HEK293cells confirmed byco-immunoprecipitation.5. Wee1B and KHDRBS3protein can be co-localized in mouse embryos byimmunofluorescence.ConclusionsWe screened a human mammary cDNA library with Wee1B as bait. Of the9newly implicated proteins, KHDRBS3was chosen for successive detailedanalysis. The immunoprecipitation and immunofluorescence analysessufficiently identified Wee1B as a novel binding partner of KHDRBS3. Proteinkinase Wee1B was interacted with KHDRBS3proteins in293cells and the bothproteins can be co-localized in the mouse embryos, suggesting that KHDRBS3proteins may regulate Wee1B in the early development of mouse embryos viaprotein and protein interaction.