Nanostructured Protein Production in E. coli

Nanostructured proteins were produced in E. coli following the method described in Torrealba et al. (9 (link)) and Thwaite et al. (12 (link)). In short, E. coli transformed with the plasmid of interest was cultured in LB with the appropriate antibiotic and recombinant protein expression was induced at OD_550nm 0.5–0.8 with 1 mM IPTG (Panreac, Barcelona, Spain). IBs were isolated after 3 h additional incubation at 37°C via enzymatic and mechanical disruption of the cells according to Torrealba et al. (10 (link)), followed by sterility monitoring (12 (link)). Purified nanoparticles, named here IB^{frg16G−VHSV}, IB^TNFα and IB^iRFP [an inclusion body made of a non-immunogenic phytochrome-based near infra-red fluorescent protein (iRFP) with the excitation/emission maxima at 690/713 nm (13 (link))], were stored at −80°C until use. Quantification was performed by western blot using an anti-His-tag antibody (Genscript, Piscataway, NJ, USA) and calculating the protein concentration from a standard curve using Quantity One software (Biorad, Hercules, CA, the USA). For flow cytometry or confocal microscopy, IB^{frg16G−VHSV} and IB^TNFα were conjugated with fluorescent Atto-488 NHS ester (Sigma-Aldrich) following manufacturer's instructions.