Methodological insights into the assembly and validation of USP22 complexes via the PQLink vector suite

USP22, as a deubiquitinating enzyme, is highly expressed in various types of cancer and promotes the progression of the cell cycle and cancer development by interacting with cell cycle regulatory factors. This study, for the first time, successfully expressed a highly active USP22 complex in vitro using a method based on the PQLink series of vectors. It was found that USP22 must form a complex with the auxiliary proteins ATXN7, ATXN7L3, and ENY2 to possess deubiquitinating enzyme activity. Through optimizing gene sequences, the construction of protein expression vectors, expression-test, overexpression and purification, we obtained a highly active USP22 complex and verified its deubiquitinating enzyme activity using Ub-AMC hydrolysis assay. In addition, the experimental results showed that the enzyme activity of the USP22 complex would be lost if any of the auxiliary proteins were missing. This study not only provides important information for understanding the mechanism of USP22 in cancer but also lays the foundation for the development of small molecule inhibitors targeting USP22, which is expected to become a new strategy for cancer treatment.