| Autor: |
Pu ZX; Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China.; Gansu Province Key Laboratory of Gene Editing for Breeding, School of Life Sciences, Lanzhou University, Lanzhou 730000, China., Wang JL; Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China.; Gansu Province Key Laboratory of Gene Editing for Breeding, School of Life Sciences, Lanzhou University, Lanzhou 730000, China., Li YY; Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China.; Gansu Province Key Laboratory of Gene Editing for Breeding, School of Life Sciences, Lanzhou University, Lanzhou 730000, China., Liang LY; Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China.; Gansu Province Key Laboratory of Gene Editing for Breeding, School of Life Sciences, Lanzhou University, Lanzhou 730000, China., Tan YT; Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China.; Gansu Province Key Laboratory of Gene Editing for Breeding, School of Life Sciences, Lanzhou University, Lanzhou 730000, China., Wang ZH; Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China.; Gansu Province Key Laboratory of Gene Editing for Breeding, School of Life Sciences, Lanzhou University, Lanzhou 730000, China., Li BL; Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China.; Gansu Province Key Laboratory of Gene Editing for Breeding, School of Life Sciences, Lanzhou University, Lanzhou 730000, China., Guo GQ; Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China.; Gansu Province Key Laboratory of Gene Editing for Breeding, School of Life Sciences, Lanzhou University, Lanzhou 730000, China., Wang L; School of Chemical Engineering Ocean and Life Science, Dalian University of Technology, Panjin 124221, China., Wu L; Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China.; Gansu Province Key Laboratory of Gene Editing for Breeding, School of Life Sciences, Lanzhou University, Lanzhou 730000, China. |
| Abstrakt: |
Ubiquitination is one of the most important post-translational modifications in eukaryotes. The ubiquitination cascade includes ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). The E3 ligases, responsible for substrate recognition, are the most abundant and varied proteins in the cascade and the most studied. SKP1-CUL1-F-Box (SCF)-type E3 ubiquitin ligases are multi-subunit RING (Really Interesting New Gene) E3 ubiquitin ligases, composed of CUL1 (Cullin 1), RBX1 (RING BOX 1), SKP1 (S-phase Kinase-associated Protein 1), and F-box proteins. In vitro ubiquitination assays, used for studying the specific recognition of substrate proteins by E3 ubiquitin ligases, require the purification of all components involved in the cascade, and for assays with SCF-type E3 ligases, additional proteins (several SCF complex subunits). Here, the Duet expression system was used to co-express E1, E2, ubiquitin, ubiquitylation target (substrate), and the four subunits of a SCF-type E3 ligase in E. coli . When these proteins co-exist in bacterial cells, ubiquitination occurs and can be detected by Western Blot. The effectiveness of this bacterial system for detecting ubiquitination cascade activity was demonstrated by replicating both AtSCF TIR1 -mediated and human SCF FBXO28 -mediated ubiquitylation in bacteria. This system provides a basic but adaptable platform for the study of SCF-type E3 ubiquitin ligases. |
