Production and Characterization of Recombinant Rat Brain-Derived Neurotrophic Factor and Neurotrophin-3 from Insect Cells
Article first published online: 23 NOV 2002
Journal of Neurochemistry
Volume 62, Issue 2, pages 471–478, February 1994
How to Cite
Negro, A., Tavella, A., Grandi, C. and Skaper, S. D. (1994), Production and Characterization of Recombinant Rat Brain-Derived Neurotrophic Factor and Neurotrophin-3 from Insect Cells. Journal of Neurochemistry, 62: 471–478. doi: 10.1046/j.1471-4159.1994.62020471.x
- Issue published online: 23 NOV 2002
- Article first published online: 23 NOV 2002
- Received March 30, 1993; revised manuscript received June 15, 1993; accepted June 22, 1993.
- Brain-derived neurotrophic factor;
Abstract: Rat brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were engineered for expression in a baculovirus-infected Spodoptera frugiperda insect cell system. The BDNF and NT-3 from the culture supernatants were purified by ion-exchange and reverse-phase chromatography to apparent homogeneity. The purification procedure yielded ∼2 mg of pure rat BDNF or NT-3 per liter of culture supernatant. A single N-terminus only was found for either secreted molecule and was analogous to that predicted from the corresponding cDNA sequence. The recombinant neurotrophins obtained were also homogeneous with regard to molecular weight and amino acid sequence. In their native conformation, the insect cell-produced rat BDNF and NT-3 molecules were homodimers consisting of 119 amino acid polypeptide chains. Thus, although the genes transfected into the S. frugiperda cells coded for proBDNF or proNT-3, the BDNF and NT-3 recovered after purification were >95% fully processed, mature protein. Mature recombinant rat BDNF and NT-3 were found not to be significantly glycosylated. Pure, recombinant rat BDNF and NT-3 promoted the survival of embryonic dorsal root ganglion neurons in the low picomolar range. Because recombinant rat BDNF and NT-3 can be obtained in large quantities, purified to near homogeneity, and are identical in amino acid sequence to the corresponding human proteins, they are suitable for evaluation in animal models.