Isolation of a Protein from Bovine Brain Which Promotes Neurite Extension from Chick Embryo Cerebral Cortex Neurons in Defined Medium

Seven-day-old chick embryo cerebral cortex neurons cultured at low density (10,000 cells/16 mm well) in defined medium in polylysine-coated wells fail to extend neurites and assume a flattened phase-dark morphology. Addition of soluble bovine brain extract promotes neurite outgrowth and rounding of the cell body (which becomes phase-bright). A quantitative bioassay was utilized to purify this neurite extension factor (NEF), based on counting the number of phase-bright neurons with processes at least equal to one cell body diameter after 20 h in culture. Using a combination of heat treatment, DEAE-cellulose chromatography, and gel filtration, an acidic protein with a native Mr = 75,000 has been purified. Upon reduction it yields subunits of Mr = 37,000. Purified fractions are active at 100 ng/ml in inducing neurite outgrowth in this bioassay.