Pet28a

The cDNA sequences encoding A. thaliana TOP1 (AT5G65620) and TOP2 (AT5G10540) were cloned into pET-28a (Agilent) in a translational frame with 6xHis N-terminal tags. Cysteine mutagenesis was performed by site-directed mutagenesis, using the wild-type cDNAs, gene-specific primers, and Phusion High-Fidelity DNA Polymerase (New England Biolabs). The PCR products were separated on agarose gels (0.8%), purified using the QIAquick gel extraction Kit (Qiagen), and cloned into pET-28a using NheI and SalI restriction enzymes (NEB) and T4 DNA ligase (NEB). The plasmids were used for the expression of TOPs in E. coli. Additional details are described in Supplemental Methods.

Cloning, expression, and purification of the Dmr1-MBP-His₆ fusion protein was preformed essentially as described previously (Puchta et al. 2010 (link)). For purification of the Dmr1-His₆ fusion protein, the codon-optimized (for yeast) sequence of DMR1 ORF (without the 26 amino acid amino-terminal mitochondrial targeting sequence) was obtained by custom gene synthesis (Generay Biotech Co.) and cloned in the pET28a vector (Novagen). The pET28a::DMR1 vector was transformed into BL21(DE3) CodonPlus-RIL E. coli (Agilent). An amount of 1 mL of the overnight culture was inoculated into 500 mL of AIM (auto induction medium, Formedium), and the cells were grown at 23°C for 36 h. Metal affinity and size exclusion protein purification procedures were performed as described previously (Malecki et al. 2008 (link)). The MBP-His₆ protein used as the negative control was expressed from the pMM41 plasmid (Mayekar et al. 2013 (link)) and purified using the same protocol. Purity and composition of the protein preparations was assessed using LC–MS–MS/MS mass spectrometry on the Orbitrap (Thermo Fisher) spectrometer at the Laboratory of Mass Spectrometry, IBB PAS. The purified proteins were stored in a buffer containing 0.5 M NaCl, 50 mM Tris-HCl pH 8, 20% glycerol.