TtClpP DNA was directly amplified from T. thermophilus HB8 DNA and cloned in to a pET41c (Novagen) expression vector using Ned I and Xho I restriction enzymes. The expressed gene contained the additional residues LEHHHHHHHH at its C terminus to allow affinity chromatography purification. Full-length ClpX DNA was first cloned into a pET28 vector using Ned I and Hind III restriction enzymes, resulting in residues MGSSHHHHHHSSGLVPRGSH added to the N terminus. While full-length ClpX was insoluble, similar to ClpX orthologs from other species, removal of its N-terminal domain (residues 1 to 54) allowed expression of a soluble ClpX construct (∆TtClpX) (48). Removal of the TtClpX N terminus was performed by GeneCust using Ned I and Hind III as restriction enzymes. TtClpP and ∆TtClpX were expressed in E. coli Bl21RIL cells following overnight expression at 20°C after induction with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). A TtClpP mutant containing an S97A mutation was constructed using the QuikChange II Site-Directed Mutagenesis Kit from Thermo Fisher Scientific.

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