Isopropyl β d 1 thiogalactopyranoside (iptg)

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.

The recombinant gene was synthesized (General Biosystems) and was cloned in pET 15b plasmid systems (Novagen). One Shot™ BL21(DE3) E. coli (Thermo Fisher) cells were transformed with the expression vector. The clone was cultured in LB broth medium, and when the optical density was 0.5 (590 nm) then Isopropylb-D thiogalactopyranoside (1.0 mM IPTG, PanReac AppliChem) was added. After 6 h at room temperature incubation, cells were collected by centrifugation and were resuspended in LEW buffer (50 mM NaH₂PO₄ and 300 mM NaCl in distilled water pH = 8.0). 1 mg/ml lysozyme and protease inhibitor (Pierce, Thermo Fischer) were added to lyses bacterial cells. Bacterial cell lysates were obtained by probe sonication (5 × 15 s pulses at 130 W, 65% amplitude, with 15 s intervals, 4 ºC) and insoluble material synthesized by centrifugation. Bacterial soluble protein lysate was loaded onto pre-equilibrated Ni-TED columns (Protino® Ni-TED, Macherey–Nagel GmbH & Co., Düren, Germany). Recombinant His-tagged protein was eluted in buffer supplemented with 250 mM imidazole. PD-10 Desalting Columns (GE Healthcare, Chicago, USA) were used to remove the imidazole and exchange buffer to PBS. Finally, saturating amounts of the recombinant protein were mixed with 200 μg o-MWCNTs, were sonicated (5 min, 2 s pulses, 65% amplitude, 4 °C), and were washed 3 times with PBS to remove the excess protein.