Degradation mechanism of important regulator AtrA mediated by ClpXP in Streptomyces roseosporus

ABSTRACT: 3×Flag-atrA strain was grown in YEME and 50 mL were collected after 24 h. Cells were resuspended in phosphate buffer (pH=7.4) (PBS) and sonicated. After centrifugation at 12000 rpm, the supernatant was transferred and incubated with FLAG affinity gel (Yeasen) at 4 °C. The FLAG affinity gel was washed three times with 1 mL binding buffer (PBS contains 10% glycerol). After centrifugation at 2000 rpm, 500 μL PBS was added, and the interaction proteins were eluted after denaturation. WT strain was used as control. Protein identification and analysis was provided by Jingjie PTM BioLab (Hangzhou, China). Proteins were expressed in BL21 with the plasmids pET-28a-3×Flag-atrA and pET28a-clpX, respectively. The purified proteins were mixed and incubated at 30 °C for 3 h. The reaction system consisted of 4 μg AtrA, 4 μg ClpX and 3 mM ATP. The elution method is the same as for the pull-down assay after FLAG affinity gel purification at 4 °C. Samples were analyzed by western blot using anti-His mouse monoclonal antibody. AtrA was expressed in BL21 with the plasmid pET-32a-atrA and incubated with cell lysate of Δprc at 30 °C for 1 h. The protein processing and pupylation analysis were provided by Jingjie PTM BioLab (Hangzhou, China).

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Streptomyces Filamentosus Nrrl 15998

TISSUE(S): Mycelium

SUBMITTER: weifeng xu  

LAB HEAD: Weifeng Xu

PROVIDER: PXD037377 | Pride | 2024-05-23

REPOSITORIES: Pride