2.1. Expression, refolding and purification of rhNGF

rhNGF was recombinantly expressed according to well established protocols which exploit the in vitro refolding and subsequent proteolytic cleavage from its precursor rh-proNGF. rhNGF was expressed in Rosetta (DE3) E. Coli cells, transformed with the pET11a plasmid containing the cDNA of human proNGF VSAR . The mutation RSKR – VSAR at furin cleavage site allowed the introduction of a specific trypsin cleavage for the preparation of mature rhNGF (US Patent 2015/0087020). Solubilization and refolding of rh-proNGF from inclusion bodies was carried out according to published protocols [40]. Mature rhNGF was obtained by controlled trypsin cleavage of rh-proNGF [41]. Optimization of the expression of rh-proNGF VSAR in minimal medium with ¹⁵N- or ¹⁵N-¹³C labeling was needed, starting from the conditions described for the recombinant mouse proNGF [41]. The best expression conditions were obtained by supplementing M9 medium with 0.4% glucose and upon induction with IPTG for 18 h at 25 °C. The integrity of rhNGF was confirmed by MALDI-MS analysis. Protein fold was characterized by biophysical techniques (DSF, FT-IR, NMR).