Jun 22, 2023
Purification and analysis of SKP1-FBXO7 complexes
- Frank Adolf1,
- Brenda A. Schulman1
- 1Max-Planck Institute of Biochemistry, Research Department Molecular Machines and Signaling
Protocol Citation: Frank Adolf, Brenda A. Schulman 2023. Purification and analysis of SKP1-FBXO7 complexes. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4z4ogmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 28, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 79610
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000282
Abstract
Protocol for the biochemical purification and analysis of SKP1-FBXO7 complexes
Guidelines
Please wear appropriate PE while performing the experiment.
Please familiarise yourself with the laboratory safety rules and guidelines and follow these while performing the experiment.
Materials
Buffer A for size exclusion chromatography (SEC):
- 25mM HEPES pH7.5 (KOH)
- 150 mM NaCl
- 1mM DTT
TB medium
Preparatory note
Preparatory note
All constructs were prepared utilizing standard molecular biological techniques and verified by sanger sequencing.
The cDNAs coding for HsFbxo7-129-398 and Skp1 preceding a second RBS were cloned into pGEX4T1 as previously described for other substrate receptor/Skp1 complexes (Schulman et al. 2000) (pGEX4T1-TEV-HsFbxo7-129-398/HsSkp1).
The cDNA coding for HsPI31-1-151 (with N-terminal TEV cleavable His8-tag) was cloned into pRSF1b (pRSF1b-His8-TEV-HsPI31-1-151).
For co-expression of Fbxo7/Skp1/PI31 complexes, both plasmids were co-transformed into E. coli BL21 Rosetta (DE3).
Molecular biological methods and protein expression
Molecular biological methods and protein expression
Grow E.coli cultures in Terrific Broth (TB) medium at 37°C.
At OD600 of 0.8, induce expression with 0.5mM IPTG.
Continue growing the E.coli culture for 16h at 18°C.
1d
Purify FBXO7/Skp1/PI31 complexes by sequential standard GST- and His-affinity chromatography.
Cleave affinity tags by incubation with TEV protease at 4°C for 16h.
1d
Further purify complexes by preparative size exclusion chromatography (SEC) in buffer A (25mM HEPES pH7.5 (KOH), 150 mM NaCl, 1mM DTT; for details please see next section) on a Superdex 200 Increase 10/300 GL column.
Pool fractions of interest, aliquoted and snap freeze in liquid N2.
Store fractions at -80°C until further usage.
HsCul1-1-410 was expressed as GST-fusion protein and purified as described previously (Hopf et al., 2022).
Analytical size exclusion chromatography (SEC)
Analytical size exclusion chromatography (SEC)
30m
30m
Analytical SEC was carried out on an ÄKTApure system (GE Healthcare) equipped with a Superdex 200 Increase 10/300 GL column (Cytiva), in buffer A (25mM HEPES pH7.5 (KOH), 150 mM NaCl, 1mM DTT).
Preincubate samples at 37°C for 10 min before loading.
Samples:
HsCul1-1-410,
HsFbxo7-129-398/HsSkp1/HsPI31-1-151
HsFbxo7-129-398/HsSkp1/HsPI31-1-151 + HsCul1-410
Apply samples (100 µl at a concentration of 45 µM) on column.
Set flow rate to 1 ml/min.
30m
Record UV absorbance at 280 nm and collect fractions of 200 µl volume.
Analyze fractions by SDS-PAGE.
Protocol references
Ref1: Schulman et al., 2000,PMID:11099048
Ref2: Hopf et al., 2022,PMID:35982156