Production and characterization of recombinant mouse neurotrophin‐3

Abstract

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 pg 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.Nerve growth factor (NGF) is the prototypical neurotrophic molecule and its important role in the survival, development and maintenance of some neuronal populations, particularly in the peripheral nervous system, has been well characterized (for review, see [I]). The tissues innervated by NGF-responsive neurons have been shown to contain and synthesize limited amounts of NGF [2-61 supporting the neurotrophic theory. This concept states that embryonic, naturally occurring cell death, involving the loss of a significant number of neurons in a given population, is regulated by neurotrophic molecules produced by the target; competition for the limited amounts of trophic factor allows the survival of only a portion of the neurons (for recent reviews, see [7, 81). The recent cloning of brain-derived neurotrophic factor (BDNF) revealed that it is structurally related to NGF [9]. The knowledge that NGF had a structurally rekated relative allowed the identification of a third member of this gene family, neurotrophin-3 (NT-3; also described as hippocampus-derived neurotrophic factor or NGF-2) [lo -151. These three neurotrophic factors are synthesized as precursors and upon proteolytic cleavage, the mature, biologically active forms are generated. They are basic proteins with a molecular mass of approximately 13.5 kDa and are characterized by six conserved cysteine residues. The cysteine residues have been shown to form three intramolecular disulfide bridges in mouse NGF [16]. In the mouse, 54 residues out of 120 are common to all three neurotrophic proteins.In view of the minute amounts of neurotrophic factors in almost all tissues and the consequently diMicult purification [18] has yielded recombinant proteins with specific biological activities several orders of magnitude lower than that of natural NGF, rendering these expression systems unattractive for the production of other species of NT. In previous experiments, only...

Purification and characterisation of a brain‐derived neurotrophic factor/ neurotrophin‐3 (BDNF/NT‐3) heterodimer

“…The procedure used here follows that described recently by Gotz et al, 1992. Briefly, DNA fragments encoding mouse wild-type (wt) neurotrophins were amplified by polymerase chain reaction (PCR; Saiki et al, 1988) using primers containing restriction sites.…”

Zinc inhibits p75NTR-mediated apoptosis in chick neural retina

“…35 Briefly, glass powder was incubated overnight in trifluracetic acid at room temperature, then beads were washed with distilled water and PBS six times each. Neurotrophins or cytochrome c were added to the activated beads at concentrations of 0.01 mg/ml and placed on ice for 1 h. Beads were washed with PBS six times and applied immediately on top of the plated cells.…”

Brain-derived neurotrophic factor prevents the death of motoneurons in newborn rats after nerve section

“…Gel foam (Spongostan, gift of K. Unsicker, Marburg, Germany) soaked in 30 ILl of PBS containing either 5 ILg of BSA (Cohn Fraction V, Sigma) or trophic factor was inserted at the site of lesion. Nerve growth factor (2.5 S) was isolated from submaxillary glands of adult male mice, as described 22 , Both recombinant mouse BDNF and NT-3 were produced in rabbit kidney cells infected with recombinant vaccinia viruses and purified as described 23 . The skin was sutured by silk (Ethikon 3-0) and the animals returned to their mothers.…”