ABSTRACT
Objective: Daidzein was efficiently purified by agar gel microspheres bonded β-cyclodextrin (AG-β-CD). Methods: Using agar as raw material, after emulsification, crosslinking, and bonding β-CD as functional group, AG-β-CD was synthesized for the purification of daidzein, and the purification process was determined and proved with mobile phase, flow rate, and loading capacity of microspheres. The structure of daidzein was identified by MS and NMR, AG-β-CD was chromatographically evaluated with daidzein, EGCG, and puerarin as the following tripartition such as difference of retention behavior on C18 reversed phase column chromatography, molecular simulation by autoDOCK4.0, and retention time curves on AG-β-CD with different contents of acetonitrile. Results: The main component of soybean isoflavone was daidzein (57.14%). The loading quantity of AG-β-CD was 1.33 mg/mL, flow rate was 2 BV/h, eluted by 2 BV of 20% ethanol, 1.33 BV of 40% ethanol, and 6-7 BV of 70% ethanol, the content was ≥ 95%, purity of daidzein (96.98%) was obtained with 97.86% yield. Chromatographic mechanism research showed that AG-β-CD had hydrophilic interaction chromatography and reversed-phase chromatography. Conclusion: AG-β-CD is capable of highly efficient purification of daidzein.
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
Aim To isolate and purify a novel plasminogen activator(PA)from Gloydius brevicaudus venom(GBV)and study characterization and biological activities of GBV-PA.Methods Affinity chromatography in Benzamidine Sepharose 6B(AC)and Lichrospher C-18 4.6/250 reversed phase chromatography(RPC)were used for isolation and purification;SDS-PAGE was used to detect molecular weight(MW);Disc polyacrylamide gel eletrophoresis was used to measure the point of isoelectric(pI);Chromogenic substrate method was used to observe the biological activities.Results A novel GBV-PA which its purification reached the homogeneity level was isolated and purified from GBV by AC and RPC;The MW of the novel GBV-PA was 3.26?104 and the pI was 5.2;The novel GBV-PA activated human plasminogen specifically and the special activity was 2.87 t-PA IU?mg-1;Moreover,our results indicated that this novel GBV-PA was a serine proteinase which had no affinity to fibrin.Conclusion A novel GBV-PA that can be isolated and purificated from GBV by AC and RPC was proved to be a serine protease and has no affinity to fibrin.