PRODUCTION AND CHARACTERIZATION OF RECOMBINANT MOUSE BRAIN-DERIVED NEUROTROPHIC FACTOR AND RAT NEUROTROPHIN-3 EXPRESSED IN INSECT CELLS

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. Yiel ds of purified neurotrophin-3 (300-500 mu g/L) were similar to levels reported for baculovirus-expressed nerve growth factor (NGF), whereas initial yields of BDNF were significantly lower (20-50 mu g/L). Improv ed production of BDNF (150-200 mu g/L) was achieved by expressing BDNF from a chimeric prepro-NGF/mature BDNF construct using the Trichoplus ia ni insect cell line, Tn-5B1-4. Examination of the distribution of B DNF protein from both the nonchimeric prepro-BDNF and the chimeric pre pro-NGF/mature BDNF viruses in Sf-21- and Tn-SB1-4-infected cells sugg ests a specific deficiency in the Tn-5B1-4 cells in processing the non chimeric 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 ch aracterized in vitro on primary neuronal cultures from the CNS and PNS .