Purification and analysis of SKP1-FBXO7 complexes

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).

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.

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.

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 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.

Record UV absorbance at 280 nm and collect fractions of 200 µl volume.

Analyze fractions by SDS-PAGE.