title: 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 micrograms/L) were similar to levels reported for baculovirus-expressed nerve growth factor (NGF), whereas initial yields of BDNF were significantly lower (20-50 micrograms/L). Improved production of BDNF (150-200 micrograms/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 non-chimeric 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.
authors.affiliation: Cephalon, Inc., West Chester, PA 19380.
authors.forename: S L
authors.initials: SL
authors.lastname: Meyer
authors.forename: D M
authors.initials: DM
authors.lastname: Lang
authors.forename: M E
authors.initials: ME
authors.lastname: Forbes
authors.forename: E
authors.initials: E
authors.lastname: Knight
authors.forename: J D
authors.initials: JD
authors.lastname: Hirsch
authors.forename: S P
authors.initials: SP
authors.lastname: Trusko
authors.forename: R W
authors.initials: RW
authors.lastname: Scott
mesh_terms: D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D019208:Brain-Derived Neurotrophic Factor; D002460:Cell Line; D005822:Genetic Vectors; D007313:Insecta / Q000166:cytology / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D009420:Nervous System / Q000166:cytology / Q000187:drug effects; D009474:Neurons / Q000187:drug effects; D020933:Neurotrophin 3; D009841:Oligonucleotides / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins; D012995:Solubility
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D020933:Neurotrophin 3; D009841:Oligonucleotides; D011994:Recombinant Proteins