Efficient ASK-assisted system for expression and purification of plant F-box proteins

Ubiquitin-mediated protein degradation plays an essential role in plant growth and development as well as responses to environmental and endogenous signals. F-box protein is one of the key components of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which recruit specific substrate proteins for subsequent ubiquitination and 26S proteasome-mediated degradation to regulate developmental processes and signaling networks. However, it is not easy to obtain purified F-box proteins with high activity due to their unstable protein structures. Here, we found that Arabidopsis SKP-like proteins (ASKs) can significantly improve soluble expression of F-box proteins and maintain their bioactivity. We established an efficient ASK-assisted method to express and purify plant F-box proteins. The method meets a broad range of criteria required for the biochemical analysis or protein crystallization of plant F-box proteins. Significance Statement Plant F-box proteins, as a key component of the SCF E3 ubiquitin ligase complex, play important roles in regulating plant growth and development during the entire plant life cycle. However, difficulties in obtaining purified F-box proteins have affected their in-depth study. In this study, we develop an effective ASK-assisted baculovirus-insect cell expression system that is suitable for the expression and purification of various F-box proteins.