Cascade subcomplexes lacking Cse1 were prepared from E. coli KD418 cells (28 (link)) co-expressing cas genes and appropriate CRISPR cassette from pCDF-based plasmids. Cascade subcomplexes containing N-terminal Strep-Tag II fused to Cse2 subunit were affinity-purified on Strep-Tactin® column (IBA) from cells grown at 37°C until OD550 reached 0.5 followed by 4-h induction with 1 mM IPTG. Purification buffers contained 100 mM Tris-HCl, pH 8, 150 mM NaCl, 5 mM β-mercaptoethanol and 1 mM ethylenediaminetetraacetic acid (EDTA). Binding buffer additionally contained 0.1 mM phenylmethanesulfonyl fluoride and Elution buffer contained 2.5 mM desthiobiotin and 1 mM (tris(2-carboxyethyl)phosphine) TCEP. Complexes were further separated using a Superdex 200 HiLoad 16/60 column (Amersham Biosciences) equilibrated by 50 mM Tris-HCl, pH 8, containing 150 mM NaCl, 1 mM EDTA and 1 mM TCEP.
N-terminally 6His-tagged Cse1 was purified by IMAC from E. coli KD418 strain transformed with an appropriate expression plasmid followed by gel-filtration on Superdex 200 HiLoad 16/60 column (Amersham Biosciences) equilibrated with 20 mM HEPES-K buffer (pH 7.5) containing 150 mM NaCl.
N-terminally 6His-tagged Cse1 was purified by IMAC from E. coli KD418 strain transformed with an appropriate expression plasmid followed by gel-filtration on Superdex 200 HiLoad 16/60 column (Amersham Biosciences) equilibrated with 20 mM HEPES-K buffer (pH 7.5) containing 150 mM NaCl.
