Production and characterization of biologically active recombinant human nerve growth factor.
Nerve growth factor (NGF) is required for the differentiation and maintenance of sympathetic and sensory neurons. In animal models, NGF prevents the death of septal and basal forebrain cholinergic neurons deprived of endogenous NGF, suggesting that NGF may be of benefit in neurodegenerative diseases of humans. However, little is known about NGF in human brain, partly because a sensitive assay for hNGF has been lacking. As a first step toward developing the tools for the study of NGF in humans, recombinant human NGF (rhNGF) was produced by expressing exon 4 of the human NGF gene in COS cells. The expression vector is driven by the adenovirus major late promoter and contains an SV40 origin of replication. NGF was secreted by transiently transfected cells. Conditioned medium was assayed with an enzyme immunoassay (EIA) that utilizes a monoclonal antibody (clone 27/21) against mouse beta-NGF, and contained 15 ng/ml of rhNGF. The rhNGF migrated as a dimer of 26-29 Kd on a gel permeation chromatography column, and stimulated neurite outgrowth and neuropeptide Y mRNA levels in PC12 cells. With optimization, the described expression system is capable of providing sufficient hNGF for research and therapeutic purposes.
Bruce
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Howard Hughes Medical Institute, Department of Medicine, Massachusetts General Hospital, Boston.
Heinrich
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D000818:Animals; D015152:Blotting, Northern; D005786:Gene Expression Regulation; D006801:Humans; D007124:Immunoenzyme Techniques; D051379:Mice; D009414:Nerve Growth Factors / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D011950:Receptors, Cholinergic / Q000502:physiology; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics
D009414:Nerve Growth Factors; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins