Characterization of the CRISPR/Cas subtype I-A system of the hyperthermophilic crenarchaeon Thermoproteus tenax - PubMed (original) (raw)

Reconstitution assays for recombinant Cas complexes Cascis and Cascade. (A) Denatured proteins Cas4, Cas1/2, and Csa1 were combined and refolded into the Cascis complex by rapid dilution. The ratio (%) between complete protein content of supernatant and pellet in different refolding buffers is shown: buffer (40 mM Tris-HCl [pH 7], 10 mM β-mercaptoethanol, 10% glycerol, 300 mM NaCl), −NaCl (buffer without NaCl), +Mg (buffer plus 10 mM MgCl2), +Ca (buffer plus 10 mM CaCl2), and +Arg (buffer plus 500 mM

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-arginine). Cascis, complete complex; pCascis, partial complex (Cas4, Csa1). (B) SDS gel electropherogram of the reconstitution assay (buffer plus 500 mM

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-arginine). Lane S, supernatant fraction (refolded complex); lane P, pellet fraction (aggregated protein). (C) Denatured proteins Cas5a, Cas3, Cas3′, and Cas8a2 and soluble purified proteins Csa5 and Cas7 were reconstituted to Cascade by stepwise dialysis. The ratio (%) between the complete protein content of the supernatant and pellet in refolding buffer (100 mM HEPES/KOH [pH 7], 10% glycerol, 300 mM NaCl, 10 mM β-mercaptoethanol; for refolding of p1CasA2, p4CasA2, and CasA2) and the addition of 5 or 30 μg of total T. tenax RNA or 30 μg of total T. tenax RNA and 10 mM CaCl2 is shown. Partial complexes: P1 (single Cas proteins) and P4 (four proteins [Cas5a, Cas3, Cas3′, and Cas8a2]). (D) SDS-gel electropherogram for the identification of the constituents of Cascade in refolding buffer under the conditions described above. Depicted are the supernatant fractions (M, protein marker; B, refolding of Cascade without addition of RNA).