Visualization of stable protein complexes with palladium , rhodium and iridium nanoparticles detected by catalytic activity in native polyacrylamide gels

Experimental Section Cloning, expression, and preparation of Pyrococcus ferritin: The open reading frame (ORF) of Pyrococcus furiosus ferritin (PfuFt) gene DSM3638 strain was codon optimized for expression in E. coli K12 (GeneOptimizer, Thermo Fisher Scientific). The PfuFt ORF in the recombinant protein is preceded by Met-Ala in order to introduce NcoI site in-frame for cloning purposes. The gene synthesis and cloning into pSFT7 plasmid were carried out by Genewiz. The coding sequence was cloned into pET28(a) bacterial expression plasmid. The protein was produced in BL21-CodonPlus (DE3)-RIL (Agilent) E. coli strain induced with 0.5 mM IPTG. Induced bacteria were collected by centrifugation and the pellet resuspended in Ft lysis buffer (FLB) (25 mM 1/2Na.HEPES, 150 mM NaCl). Bacteria were lysed by sonication, centrifuged and the clarified lysate was heated for 15 min to 80 °C in order to denature mesophilic proteins that were not removed by centrifugation. Cleared supernatant was further treated with DNaseI (10 μg/ml) for 3 h at 37 °C in order to remove bacterial DNA. DNaseI was removed by second heat treatment (10 min at 90 °C). PfuFt was concentrated from clarified heat-treated samples by filtration through Amicon Ultra-2 (Ultracel – 100K membrane with exclusion limit 100 000 NMWL) (Millipore). Low molecular weight contaminants were removed by size exclusion chromatography with PD MiniTrap G-25 (GE Healthcare) equilibrated with FLB. The protein assembly and purity were analyzed by native and denaturing polyacrylamide gel electrophoresis.