Study On Separation And Purification Of Nerve Growth Factor From Venom Of Chinese Cobra By Chromatography

Nerve growth factor (NGF) is a protein necessary for the maintenance and development of sympathetic and sensory neurons. It can not only promote the fission of neuron but also determine the growth direction of neurons. It also plays an important physiological role in central nervous system. So therapy with NGF for neurological disorders, such as Alzheimer's disease, Parkinson's disease, etc., is valuable.To identify a simple, fast and high efficient method for separation and purification of nerve growth factor (NGF) from venom of Chinese Cobra, the combination process of several different kinds of chromatographic media was studied. According to the properties of NGF and the character of different chromatographic media, a novel two-step chromatographic purification method, consisting of chromatography of crude venom on DEAE-Sepharose F.F. anion-exchange medium followed by a size exclusion on Sephadex G-50, is presented in this paper. The DEAE-Sepharose F.F. chromatographic column was 20cm×3.5cm i.d. and first eluted with the 50mmol/L Tris-HCl (pH 8.5) for 50 min, and separately followed by the elution with a linear gradient of 0-200mmol/L NaCl containing 50mmol/L Tris-HCl (pH 8.5) for 190min and that with 200mmol/L NaCl containing 50mmol/L Tris-HCl (pH 8.5) for 60min. The flow-rate of mobile phase was 6.1ml/min. The Sephadex G 50 column was 150 cm×3.5cm i.d. and eluted with 50mmol/L PBS (pH 7.5) for 1250min. The flow-rate of elution solution was 2.4ml/min. Through this two-step chromatographic purification process, the obtained NGF was proved to be homogeneous on SDS-PAGE and its molecular weight was estimated to be approximately 14000, which was consistent with that reported in literatures; and on Shim-pack VP-ODS reversed-phase high-performance chromatographic column(4.6 mm i.d.×150mm), its purity was about 97.5%. The total protein recovery of this purification method is 0.51% and the obtained NGF had the activity of eliciting neurite outgrowth from chick embryonic dorsal root ganglia. This two-step chromatographic process is simple and high efficient, and the indexes of aim prodction are no less than the traditional method's, it can be used to isolate and purify NGF from venom of Chinese Cobra in large-scale.Peng Wu (Biochemical Engineering) Directed by Bian Liujiao...