ABSTRACT
Nerve growth factor(NGF) was firstly discovered as a member of neurotrophin family,and the research and development of NGF has been lasting more than fifty years since its discovery.To this end,a two-step and high –yield chromatographic method which consists of cation ion-exchange chromatography and reversed-phase chromatography was reported to isolate recombinant human beta nerve growth factor(? –NGF) secreted by constructed Chinese hamster ovary cells(CHO/dhfr-) from the culture media.Through the process of purification,the purity of protein which was determined by SDS-PAGE and RP-HPLC has reached to 95%,and the recovery of ? –NGF routed by RP-HPLC could be 70%.Furthermore,the biological activity of final purified protein evaluated by PC12 cells and dorsal root ganglia(DRG) exhibited the same performance as the standard protein of ? –NGF bought from Sigma,which indicated that there is no loss of biological activity through the isolation process.The conclusion suggested that an economical isolation method of recombinant human ? –NGF could be practiced on the industrial process of purification.
ABSTRACT
Though prokaryotic cells could hardly express recombinant human beta nerve growth factor (rhNGF-?) with a proper three-dimensional conformation, using of E. coli as a host for industrial production of rhNGF-? is controversial. Recombinant human beta NGF was expressed in E. coli and was refolded in vitro. The isolated products was shown to be consistent with those expressed and secreted by CHO cells in biochemical characters by SDS-PAGE, RP-HPLC, mass spectrometry, N terminal analysis and bioassay determined using DRG and PC12 cells. The products can be acquired with 95% purity, 1.8 ng/U biological activity from both expression system,which remain invariable biological activity when lyophilized in excipient and store at 37℃?2℃, RH 75%?5% for 3 months. Moreover, the product refolded from inclusion bodies of E. coli shows the predominance in homogeneity and the lower cost.