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Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe²⁺ had an activating effect on the ribonuclease activities of two isoforms while Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Ag⁺, Cu²⁺, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.
Subject(s)
Angelica sinensis , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Hydrogen-Ion Concentration , Kinetics , Molecular Weight , Protein Isoforms , TemperatureABSTRACT
Objective To investigate the optimal isolation and purification conditions of flavonoids in Gnaphlium affine Thunb. and the content of flavonoids in different parts of Gnaphlium affine Thunb. was measured. Methods The separation and purification abilities of D-101 macroporoue adsorbing resins for flavonoids in Gnaphlium affine Thunb. were studied with adorption and desorption as index. The static adsorption and dynamic adsorption methods were used to analyze the effects of static saturated adsorption, static elution rate, sample concentration, sample pH value, eluent concentration and amount of eluent. The flavonoids concentrations were determined with rutin as standard. Results The D-101 macroporous adsorption resin had good effect on the separation and purification of flavonoids from Gnaphlium affine Thunb.; The optimal conditions for purification were: sample concentration 1.0 mg/ml, sample pH=4, 60％ ethanol as desorption solvent, washing flow 2 BV/h, with these parameters. With such condition, the purity of flavonoids in Gnaphlium affine Thunb. was 60.92％; The content of flavonoids in Gnaphlium affine Thunb. showed the highest content was in the stem, the second in the flower, and the least in the root. Conclusions The purification of flavonoids from the D-101 macroporous adsorption resin increased in the best purified separation conditions and the content of flavonoids was the highest in the leaf.
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To prepare the human placental growth factor 2 (PIGF-2 ),human PIGF-2 gene was cloned into pPIC9K vector to construct the recombinant pPIC9K-PIGF-2 vector. Linearized recombinant pPIC9K-PIGF-2 was transformed into Pichia pastoris by electroporation. YPD-Geneticin plate was used to screen geneticin hyper-resistant colonies. The positive colonies were verified by PCR. Results of SDS-PAGE and Western blot showed that recom-binant human PIGF-2 was expressed after being induced by methanol. Using its characteristic heparin binding, recombinant human PIGF-2 was successfully purified by heparin affinity column chromatography.
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To optimize the purification process of pigments from Coreopsis tinctoria with macroporous resins by establishing second regression model with response surface methodology. The experiment showed that XDA-7 resin had the best purification effect for pigments from C. tinctoria. The optimal absorption conditions for pigments from C. tinctoria were determined as follows： concentration of pigments solution 2.7 g•L⁻¹, flow rate 6 mL•min⁻¹, pH 6. Under these conditions, the absorption rate of pigments was up to 94.16%. Optimal desorption conditions were as follows： ethanol concentration 64%, flow rate 5 mL•min⁻¹, elution dosage 4 BV. Under these conditions, pigment desorption rate was as high as 98.72%.
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Objective To explore the effect of purification on monoclonal antibody (MAb) against PGRP by Protein A-Sepharose affinity chromatography, and to provide some based data for the purification of other antibody using the same method. Methods The ascites which include MAb was purified by Protein A-Sepharose affinity chromatography. The purity and activity of MAb was tested by SDS-PAGE and ELISA. The biological function was identified by flow cytometer and immunohistochemistry. Results The average concentration of protein in ascites before purification is 23.62 mg/ml. Before and after purification, the total protein is 148.79 mg and 146.67 mg, respectively. The recovery coefficient of protein is 98.58%. The concentration of MAb in ascites is 5.21 mg/ml averagely. The MAb purity is more than 95 %. The immunoactivity of purified antibody is higher than that of unpurified antibody. Conclusion The purity of MAb against PGRP purified by Protein A-Sepharose affinity chromatography is very high. The immunoactivity of purified antibody is higher than that of unpurified antibody. So the ProteinA-Sepharose affinity chromatography is a rapid, convenient and reliable method for the purification of MAb Against PGRP.
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Objective The experiment aims at probing the best condition of the isolation and purification of rat islets.Methods The islets were isolated from rat pancreas by hepatopancreatic duct perfusing with collagenase and purified with Ficoll 400 discontinuous density gradient centrifugation.Then the purified islets were subjected to histological staining,electron microscopy and radioimmunoassay for identification of specificity and viability.Results The histological staining revealed that the viability and the purity of the purified islets were above 95％and 85％respectively.Electron microscopy showed that the purified islets were morphologically intact with clear membrane and plenty of secreting granules.Radioimmunoassay demonstrated the secreted insulin concerntration between low-glucose groups and high-glucose groups varied significantly,which verified the good function of the islets.Conclusion Hepatopancreatic duct perfusing with collagenase is a good method for digestion.There aye many factors that influence the quantity and quality of the acquired islets,such as the completed expansion of pancreas,the concentration and viability of collagenase and the digested time,etc.
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[Aim] To gain the fusion protein purified GST-AEP.[Methods] The protein GST-AEP was expressed in E.Coli-DH5? as a fusion protein induced by IPTG.The protein was a kind of inclusion body.The purifying and refolding to inclusion body were optimized.The purity of GST-AEP was identified by 12% SDS-PAGE and thin-layer scanning analysis.The quantitation of the fusion protein GST-AEP was done with BCA Protein Assay.[Results] Purity of GST-AEP was higher than 90% and concentration was about 0.163?g/?l.[Conclusion] The fusion protein was highly purified and the method of fusion protein purification from the inclusion body was developed,which was the basis for further study on AEP.
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Objective To simply the method of how to obtain cultures rich in astrocytes from SD rat cerebral cortex which could be utilized in vitro experiments.Methods The neonatal rat cerebral cortex was made into suspension by mechanical dissociation,and then reduced other cells by differential velocity adherent technique,shaking in orbital shaker and passage of cultured cells.After purification,the cultured cells were identified by double immunofluorescence staining and SABC.Results We successfully obtain cultures rich in astrocytes and the proportion of astrocytes were more than 95%.Conclusion The method described above was reliable in obtaining the high purity astrocytes from neonatal rat cerebral cortex and double immunofluorescence staining was more vivid and direct.
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Aim To purify Phospholipid-binding anticoagulation protein(PBAP) from Agkistrodon halys Brevicaudus Venom and study the biochemical characterization.Methods The Phospholipid-binding anticoagulation protein was purified from Agkistrodon halys Brevicaudus Venom by Cation ion exchange chromatography on CM Sephadex C-25 and negative ion exchange chromatography on DEAE Sepharose CL-6B,gel filtration on Sephacryl S-200 and Sephadex G-75 chromatography.Its anticoagulant activities in vitro were assayed by activated partial thromboplastin time(APTT);its molecular weight was calculated by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and its isoelectric point was estimated by the isoelectric focusing electrophoresis.Binding experiments of anticoagulation protein to phospholipids vesicles were performed with thinlayer chromatography.Results A kind of protein was purified from Agkistrodon halys Brevicaudus Venom which was able to prolong APTT.The SDSPAGE showed that it was dimer and its molecular weight was 24.0?10~3 under non-reducing condition and 14.6?10~3 under reducing condition.The isoelectric point was pH 5.2 by the isoelectric focusing electrophoresis.Having arginine ester-hydrolyzing enzyme and binding Phospholipid activify,its effect on APTT was activity stronger with concentration increasing.Conclusion It is a successful method of the purification of Phospholipid-binding anticoagulation protein from the Agkistrodon halys Brevicaudus Venom.
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This paper reviews the recent advances in recombinant expression and purification of defensins, including the choice of host cells, vectors and expression strategies in prokaryotic and eukaryotic cell ex- pression systems, as well as the status of purification processes. By summarizing the problems existed in the production and clinical applications of defensins, the authors here also pointed out the research directions for defensins, and conceived the prospects for its exploitation in the future.
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Objective The experiment aims at probing the best condition of the isolation and purification of rat islets. Methods The islets were isolated from rat pancreas by hepatopancreatic duct perfusing with collagenase and purified with Ficoll 400 discontinuous density gradient centrifugation.Then the purified islets were subjected to histological staining,electron microscopy and radioimmunoassay for identification of specificity and viability. Results The histological staining revealed that the viability and the purity of the purified islets were above 95% and 85% respectively.Electron microscopy showed that the purified islets were morphologically intact with clear membrane and plenty of secreting granules.Radioimmunoassay demonstrated the secreted insulin concerntration between low-glucose groups and high-glucose groups varied significantly,which verified the good function of the islets.Conclusion Hepatopancreatic duct perfusing with collagenase is a good method for digestion.There are many factors that influence the quantity and quality of the acquired islets,such as the completed expansion of pancreas,the concentration and viability of collagenase and the digested time,etc.