Production and characterization of recombinant mouse neurotrophin-3
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Neurotrophin-3 (NT-3) is a neurotrophic-factor that has recently been cloned on the basis of its structural similarity to other members of the nerve growth factor (NGF) gene family. In order to produce this protein, a recombinant vaccinia virus containing the coding region for mouse NT-3 was constructed. Conditioned medium harvested from cells infected with the recombinant vaccinia virus contained biologically active NT-3 that was purified by chromatography on controlled-pore glass and reversed-phase and gel-filtration high-pressure liquid chromatography. Approximately 200 micrograms purified NT-3 was obtained from a single cell-factory flask (1.6 1 conditioned medium). N-terminal protein sequencing showed that the mature factor was processed from the precursor at the expected site and its amino acid composition agreed with that predicted from the DNA sequence. The biological activity of the recombinant protein was tested on dissociated neurons prepared from chick embryos. Using spinal sensory neurons, the concentration of purified recombinant NT-3 allowing half-maximal survival was determined to be 25 pg/ml.
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