HyperISGylation of Old World Monkey ISG15 in Human Cells

Pattyn, Els; Verhee, Annick; Uyttendaele, Isabel; Piessevaux, Julie; Timmerman, Evy; Gevaert, Kris; Vandekerckhove, Joël; Peelman, Frank; Tavernier, Jan

Co-immunoprecipitation procedure 2610 6 Hek293T cells were transfected with the indicated expression vectors. Cleared lysates (modified RIPA lysis buffer: 200 mM NaCl, 50 mM Tris-HCl pH 8, 0,05% SDS, 2 mM EDTA, 1% NP40, 0,5% DOC, Complete TM Protease Inhibitor Cocktail (Roche)) were prepared two days after transfection. The samples were incubated with anti-FLAG M2 affinity gel (Sigma). After immunoprecipitation, SDS-PAGE and Western Blotting, interactions were detected using an anti-V5 antibody (Invitrogen) Production and purification of Hu and AgmISG15 proteins Mature ISG15 (with removed C-terminal peptide) was cloned in the pTYB1 vector and purified with the IMPACT TM procedure (Intein Mediated Purification with an Affinity Chitin-binding Tag) (New England Biolabs) When the cultures reached an OD600 of 0.6, isopropyl b-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.3 mM, and were grown at 15uC overnight. The next day, cells were removed from the medium by centrifugation. The cell pellets were resuspended in ice-cold cell Lysis Buffer (20 mMTris pH 7.5, 500 mM NaCl) and disrupted mechanically by sonication. After removal of cell debris by centrifugation, the clarified lysate was loaded onto a chitin column (equilibrated with 10 volumes of the column buffer (20 mM Tris, 500 mM NaCl, 1 mM EDTA, pH 9.0). Three bed volumes of the cleavage buffer (20 mM Tris, 1 mM EDTA, 50 mM DTT, pH 9.0) were loaded onto the column, and incubated overnight. The ISG15 protein was eluted from the column using twice 4ml column buffer and dialysed against 3 times 5 L PBS overnight.