Ernest Knight

Production and Characterization of Recombinant Mouse Brain‐Derived Neurotrophic Factor and Rat Neurotrophin‐3 Expressed in Insect Cells

Abstract: Bioactive brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 were produced using the baculovirus expression system and purified to homogeneity using ion‐exchange and reversed‐phase chromatography. Yields of purified neurotrophin‐3 (300–500 μg/L) were similar to levels reported for baculovirus‐expressed nerve growth factor (NGF), whereas initial yields of BDNF were significantly lower (20–50 μg/L). Improved production of BDNF (150–200 μg/L) was achieved by expressing BDNF from a chimeric prepro‐NGF/mature BDNF construct using the Trichoplusia ni insect cell line, Tn‐5B1‐4. Examination of the distribution of BDNF protein from both the nonchimeric prepro‐BDNF and the chimeric prepro‐NGF/mature BDNF viruses in Sf‐21‐and Tn‐5B1‐4‐infected cells suggests a specific deficiency in the Tn‐5B1‐4 cells in processing the nonchimeric precursor. In addition, the vast majority of the BDNF protein at 2 days after infection was intracellular and insoluble. N‐terminal amino acid sequencing of purified recombinant BDNF and neurotrophin‐3 demonstrated that the insect cells processed their precursors to the correct N‐terminus expected for the mature protein. Bioactivity was characterized in vitro on primary neuronal cultures from the CNS and PNS.