The tumor suppressor LZTR1: Its expression, purification and characterization

Leucine-zipper transcription regulator 1 (LZTR1) serves as a tumor suppressor gene that is highly mutated and has been implicated in the pathogenesis of diverse cancers and developmental disorders. The LZTR1 protein is a member of the BTB-Kelch superfamily and functions as an adaptor that enables the recognition and recruitment of RAS proteins, which are then targeted for ubiquitination by the Cullin3-RING ligase E3 (CRL3) complex. Understanding the assembly mechanisms, substrate recognition patterns, and pathological processes associated with RAS ubiquitination mediated by LZTR1-CUL3 is critical. In this study, we report the expression, purification, and characterization of the LZTR1 to investigate its molecular mechanisms. Our findings demonstrate that the BacMam expression system significantly enhances LZTR1 production and simultaneously improves its stability and solubility. Furthermore, the existence of CRL3 contributes to stabilizing and homogenizing LZTR1 through facilitating the formation of the complex. Additionally, we identified MRAS as a substrate that binds tightly to LZTR1, in contrast to RIT1 and HRAS. Successfully expressing and purifying the full-length CRL3^LZTR1-MRAS complex provide a foundation for future structural and functional investigations and offer a potential approach for exploring other BTB proteins with similar characteristics.