Production and Purification of Recombinant Proteins
The following constructs for expression of glutathione S-transferase (GST) fusion proteins, namely, were used: GST-USP8542-1080 (kindly provided by Dr. E. Martegani, University of Milano-Bicocca, Italy), GST-USP81_438, and GST-USP81-m (kindly provided by Dr. S. Urbe, School of Biomedical Sciences, Liverpool, U.K.), and GST-USP8542–660. GST-USP8542_1Q80 fusion construct contains the second SH3-binding motif (amino acids 666-731) of USP8 while GST-USP8542–660 contains the Ras-GRF1-binding domain; GST-USP81_438 and GST-USP81_133 fusion constructs contain the USP8 microtubule interacting and trafficking/transport (MIT) domain (amino acids 1133). Protein production, including GST alone to be used as a positive control, was induced in Escherichia coli DH5 alpha cells grown in Luria-Bertani medium (Life Technologies, Carlsbad, CA) supplemented with ampicillin, using 0.1 mM isopropyl-1-thio-beta-D-galactopyranoside (Sigma-Aldrich) for 4 h at 25°C. The bacteria were lysed by sonication in ice-cold 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 5% (v/v) glycerol, 0.5 mM dithiothreitol (DTT), plus a complete protease inhibitor cocktail (Sigma-Aldrich) and 1% Triton X-100 (final concentration).canadianneighborpharmacy.com
The lysates were clarified by centrifugation at 25 000 X g for 15 min at 4°C, and the GST fusion proteins were affinity-purified by batchwise incubation with glutathione-Sepharose beads (GE Healthcare) at 4°C according to manufacturer’s instructions. The proteins were eluted with 20 mM glutathione (Sigma-Aldrich) in 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5 mM DTT. GST-USP8542 Ш80 was then dialyzed at 4°C against 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5 mM DTT, and 10% (v/v) glycerol; GST-USP8542–660, GST-USP81–438, and GST-USP81–133 were dialyzed against 80 mM Pipes, pH 6.9, 1 mM MgC^, 1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM DTT, and 10% (v/v) glycerol. Protein concentration was determined using the Bio-Rad DC protein assay. The purity of the GST-fused proteins was evaluated by SDS-PAGE and Coomassie Blue staining. If necessary, the purified proteins were concentrated with Centricon (Millipore, Billerica, MA). GST-USP8542–660, GST-USP81–438, and GST-USP81–133 were ultracentrifuged with the TLA-100 rotor (Beckman Instruments, Palo Alto, CA) at 100 000 X g for 45 min, 4°C, before use.
Pull-down and Binding Assays
For pull-down experiments, 1% Triton X-100-soluble protein lysate (180 ig) from freshly isolated spermatids was incubated with 5 ig of purified GST-USP8542 1080 or GST alone for 2 h at 4°C, followed by 1 h of incubation with glutathione-Sepharose beads. The beads were washed three times with washing buffer (50 mM Tris, pH 7.8, 100 mM NaCl, and 1 mM EDTA containing 0.1% Triton X-100, final concentration, and 10 ig each of leupeptin and aprotinin per milliliter) and once with washing buffer minus Triton X-100 before elution in SDS-PAGE sample buffer. Equal amounts of samples were resolved by SDS-PAGE and analyzed by Western immunoblotting.