Isolation and Purification of Enzymatic Hydrolysates of Yam(Dioscorea opposita Thunb.)Proteins

Using yam （Dioscorea opposita Thunb. ） as the experimental material, enzymatic hydrolysates of yam proteins were prepared with alkaline protease, which were then isolated and purified by cellulose DE-52 anion exchange chromatography Sephadex G-50 gel chromatography. According to the results, four absorp- tion peaks were obtained by cellulose DE-52 anion exchange chromatography, and fractions at each absorption peak were collected. Specifically, the reducing ability of four peaks demonstrated a descending order of peak 2 （P2） 〉 peak I （ P1 ） 〉 peak 3 （P3） 〉 peak 4 （P4）. By Sephadex G-50 gel chmmatography, P1 and P2 were isolated and purified with distilled water and Tris-HC1 buffer, and two absorption peaks were obtained, respectively.

A Simple Method for the Isolation and Purification of 2,4-Dihydroxy-7-Methoxy-2H-1,4-Benzoxazin-3(4H)-One (DIMBOA) from Maize (Zea mays L.) Seedlings

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3（4H）-one （DIMBOA）, the dominant benzoxazinoid hydroxamic acid in maize （Zea Mays L.）, serves as important factors of resistance against insects and microbial diseases, allelochemicals used in competition with other plants. In this paper, a novel and simple method for the isolation and purification of DIMBOA from maize seedlings was developed. Frozen shoots from 7-d-old maize seedlings （1 000×g） were firstly defrosted and then were directly homogenized and extracted with ethyl acetate. The macerate was allowed to stand at room temperature （25±2）°C for 1 h to allow enzymatic release of DIMBOA from DIMBOA-glucoside. Then the ethyl acetate phase was filtered, dried and evaporated to dryness. The resulting light-tan, semicrystalline residue was stored at -20°C for 24 h. Upon recrystallization from acetone-hexane, a relative higher yield （0.58 g） of pure DIMBOA crystals was obtained compared with the yield afforded by Woodward methodology （0.26 g）.

Isolation, Purification and Cryopreservation of Cells from Neonatal Bovine Testis

The development and application of spermatogonial stem cell technology have an important significance in animal cloning, preservation of endangered species and spermatogenesis research. In this study, the seminiferous epithlium cells were isolated and purified, the ceils were cryopreserved after identification, and the effects of different purification and cyopreservation methods on bovine testicular cells were studied. The results showed that there were spermatogonial stem cells and sertoli cells in the neonatal bovine seminiferous tubules, differential adherent selection methods could effectively separate these two cell types. Spermatogonial stem cells were positive after AKP, C-kit, and OCT-4 identification; sertoli ceils were positive after oil red O and vimentin identification. Frozen stock solution supplemented with 10% DMSO had the best effect in spermatogonial stem cell cryopreservation, while fxozen stock solution supplemented with 10% of ethylene glycol and 0.1 mmol. L^-1 trehalose had the best effect in sertoli cells crvooreservation.

Purification and Analysis of Abscisic Acid-Specifically-Inducible Proteins from Rice Callus

Two ABA-specifically-inducible proteins from rice callus were isolated and purified by precipitation with 65-100% saturated （NH4）2SO4, followed by the DEAE-sepharose, TSK-gel, and two-dimension electrophoresis. Iso-electric points （pl） of the proteins with the same molecular mass （24.5 kD） were 6.1 and 6.9, respectively. The Western blot analysis indicated that the proteins expressed in different tissues were obviously different. The A1 （pl 6.1） protein was only detected in calli treated with ABA and seed embryos （SE）. However, the A2 （pl 6.9） protein was found not only in the calli treated with ABA and SE, but also in the white dry callus. Thus it suggested that the two proteins might play some important roles in the processes of seed embryo （or somatic embryo） formation.

Isolation and Purification of Unstable Iridoid Glucosides from Traditional Chinese Medicine by Preparative High Performance Liquid Chromatography Coupled with Solid-phase Extraction

An efficient preparative method was successfully developed for isolation and purification of unstable components from medicinal plant extracts, using a combined method of preparative high performance liquid chro-matography(HPLC) and solid-phase extraction(SPE). The aim of this study was to obtain an effective method with high preparative efficiency and importantly to avoid the transformation of unstable compounds. The preparative HPLC system was based on an LC/MS controlled four-channel autopurification system. The SPE method was performed with a C18 packing material to trap the target compounds and to remove the acidic additive derived from the mobile phase. Using this method, the unstable iridoid glucosides(IGs) as model compounds were successfully isolated and purified from the extract of Hedyotis diffusa Willd. Six IGs(including one new minor IG) and one nucleotide compound were simultaneously obtained, each with a purity of 91% as determined by HPLC. The structures of the isolated compounds were identified by UPLC/Q-TOF MS, UV, 1D and/or 2D NMR. It was demonstrated that the combination of preparative HPLC with SPE is a versatile tool for preparative purification of unstable compounds from complex natural products.

Isolation and Purification of Myxobacteria in Chengdu

[ Objective] To lay the basis for further studying bioactive substances in myxobacteria. [ Method] A total of 35 samples including fresh water, mud, rabbit dung, sheep dung, weathered rock and rotten wood were collected from districts and counties of Chengdu. After pretreatment, the myxobacteria were isolated and purified using different methods. Vegetative cells, myxospores, fruiting body and colony morphology of these myxobacteria strains were observed. E ResultJ Forty-two myxobacteda strains were isolated and purified. They were classified into seven genera including Myxococcus, Angiococcus, Chondromyces, Sorangium, Melittangium, Stigmatella and Archangium. The plating efficiency of myxobacteria was highest in the fresh water samples. [ Conclusion] Abundant myxobacteria resource is found in fresh water.

Isolation and Preparative Purification for Ginkgolides A and B

In this paper a simple preparative method for isolation and purification of ginkgolides A and B was developed,As starting material,a commercially available standardized ginkgo extract (EGb761,containing 24% flavonoid and 6% terpene trilactones) was used,After a pretreatment step,optimized by the uniform design method ,the concentrated intermediate extract with high content of GA and gb(+90%) was separated into the individual terpenes by preparative liquid chromatography eluted with petroleum ether-ethylacetate,Analysis of products was carried out by means of HPLC-ELSD(evaporative light -scattering detector),The results show that ginkgolides A and B are obtained in higher yield and better purity.

STUDIES ON THE ISOLATION PURIFICATION AND CHARACTERIZATION OF THE FUCOIDAN-GALACTOSAN SULFATE (FGS) FROM LAMINARIA JAPONICA

FGS, isolated from the water solution of enzymolyzed laminaria japonica, is a mixture of acid heteroglycans. Four fractions F1, F2, F3, and F4 were obtained from FGS by ion exchange chromatography. After further purified by gel filtration chromatography on a sepharose 2B and 6B column, we obtained F9, F10, F11, and F12. They showed single band when identified by electrophoresis. The molecular weight of F9, F10, F11, and F12 was estimated to be 216, 120, 138 and 140KD respectively. They containedа-glucosidic bond by IR and 1H-NMR analysis. The typical absorption peaks of these polysaccharides were showed in UV and IR spectra. These polysaccharides contained rha, fuc, man, gal, and uronate when identified by paper chromatography (p.c) and gas chromatography (GC). The molar ratio of these sugars was also assayed.

The Isolation, Purification and Identification of Fumitremorgin B Produced by Aspergillus fumigatus

Twenty-six strains of Aspergillus fumigaius were screened for toxigenicity for fumitremorgins A and B. Twenty-three of 26 strains can produce fumitremorgin B in rice medium determined by TLC and HPLC, and no fumitremorgin A was detected. The strains of no.C4104 and no. 3656 were inoculated onto 5 kg of rice media and incubated in a modified procedure. Finally, 4.0 g of fumitremorgin B was obtained after extraction and purification by modified methods, and was confirmed by TLC, HPLC, spectral analysis together with other physicochemical analysis. This is the first report of the preparation of fumitremorgin B in China.

Isolation,Purification and Immunomodifying Activity of Polysaccharides from Phellinus igniarius

Phellinus igniarius polysaccharides were obtained by hot water extraction of mushroom fruit bodies followed by ethanol precipitation,and further purified by anion exchange and gel filtration chromatography.Immuno-enhancing activity was evaluated by determining the effect of different polysaccharide fractions on mouse spleen lymphocyte proliferation in vitro.Three crude polysaccharide preparations,designated PIP30,PIP60 and PIP80 according to the percentage concentration of ethanol required for precipitation from hot aqueous extracts,promoted lymphocyte proliferation to varying degrees.Purification of polysaccharide present in PIP30 and PIP60 was carried out using anion exchange chromatography.Proliferation rates in samples treated with 200 μg/mL of fractions P30U,P30W and P30S1(obtained following anion exchange chromatography of PIP30) were approximately 5-,4-,and 3.5-fold higher,respectively compared with negative controls.Similarly,marked increases(3.4-and 2.8-fold) in cell proliferation rates compared with negative controls were observed with 200 μg/mL concentrations of P60W and P60S2(obtained following anion exchange chromatography of PIP60),respectively.Two purified polysaccharide preparations,P60W1-1 and P60S1-1,were obtained following gel filtation chromatography of fractions P60W1 and P0S1,respectively.Fraction P60W1-1(10-100 g/mL) enhanced splenocyte proliferation 0.5-1.4-fold compared with negative controls,whereas no effects were observed with P60S1-1.

Prokaryotic expression and purification of fibronectin leucine rich transmembrane protein 3 C-terminal domain proteins in rats

BACKGROUND： Studies have suggested that fibronectin leucine-rich transmembrane protein 3 （FLRT3） is related to injury and regeneration of the nervous system. However, the expression and biological characteristics of these proteins remain poorly understood. OBJECTIVE： To obtain FLRT3 C-terminal gene fragments, to effectively express and purify the target proteins. DESIGN, TIME AND SETTING： An observational study of cellular and molecular biology was performed at the laboratory of Histology and Embryology in Xiangya School of Medicine, Central South University between October 2007 and June 2008. MATERIALS： Three Sprague Dawley adult rats were used to extract total RNA from rat brains. The pGEX4T3 and Escherichia coil （E. coil） JM109 were purchased from Promega. E. coil BL21 was provided by Novagen. METHODS： FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were amplified using RT-PCR technique from rat total RNA. The amplified products were cloned into the expression vector pGEX4T3. A recombinant expression vector was then constructed and introduced into E. coil BL21. IsopropyI-D-thiogalactopyranoside was applied to induce expression of recombinant GST fusion proteins, followed by isolation, purification, and renaturation of inclusion bodies that comprised recombinant proteins. Finally, the purified recombinant protein was obtained. MAIN OUTCOME MEASURES： Determination of FLRT3 C-terminal DNA sequence; expression of target proteins was assayed by SDS-PAGE electrophoresis; purified recombinant protein was identified with Western blot methods. RESULTS： FLRT3 protein coding C-terminal DNA fragments, at a length of 786 bp, were successfully harvested through RT-PCR amplification, and were then clones into the prokaryotic expression vector pGEX4T3. The results of the sequence were consistent with the known gene sequence. SDS-PAGE analysis demonstrated that there was a specific protein band in the recombinant GST fusion proteins at a relative molecular mass of 56,600. The recombinant protein was observed in the inclusion body, and highly purified recombinant proteins were obtained through a series of methods, such as rinsing, purifying, dissolving, and renaturing. CONCLUSION： From adult Sprague Dawley rats, FLRT3 C-terminal gene fragments were successfully cloned and shown to be effectively expressed in E. coil BL21. Moreover, highly purified GST fusion proteins were obtained.

Isolation and Purification of Bovine Colostrum sIgA and IgG

To further utilize bioactive substance such as bovine colostrum sIgA and IgG, sIgA and IgG were isolated and purified simultaneously by salting out, ultrafiltration and gel chromatography, etc. The analysis of results were showed quantitatively by nonhydrogenized SDS-PAGE, and quanlities of sIgA and IgG were respectively detected by Western Blot. The results showed that the purity and yield of bovine colostrum sIgA were 85.3% and 42.8%, respectively, while the purity and yield of bovine colostrum IgG were respectively 97.2% and 64.4%. This preparative method provides theoretical and experimental foundation for sIgA industrial production.

Isolation and Purification of Antibacterial Peptides from Mussel

[ Objective ] This study aimed to extract antibacterial peptides from mussel. [ Method ] Blue mussels were used as raw materials for direct extraction of antibacterial peptides by using O. 5 % acetic acid, and the antibacterial peptides were isolated and purified by Sephacryl S-100 polyacrylamide gel chromatography. The fractions were collected for measurement of antibacterial activity and minimum inhibitory concentration for a variety of bacterial species by filter paper diffusion assay. Molecular weight of the antibacterial peptides was determined by SDS-PAGE. Variation of antibacterial activity of antibacterial peptides was measured at 100 ~C under conditions of different processing time and different pH. [ Result] The O. 5% acetic acid was used for crude extraction of antibacterial peptides as extrac- tion solution and led to relatively high extraction efficiency. By using Sephacryl S-100, the antibacterial peptides could be purified as a single substance. The isola- ted and purified antibacterial peptides of mussel had relatively strong antibacterial properties with molecular weight of 5 908, showing heat-resistance acid-alkaline resistance. [ Conclusion] This study laid the theoretical foundation for large-scale production of antibacterial peotides.

AN EFFICIENT METHOD FOR ISOLATION AND PURIFICATION OF THE PANCREATIC ISLETS

On the base of the one step, operator independent method which was set up by Christophe A.E., the pancreas was infused with cold University of Wisconsin(UW) solution for the preservation, digested by the collagenase P, circuited with HBSS+5%fetal calf serum(FCS)+10mmol/L Hepes solution, and separated with the stainless steel mesh. The number of the collected islets were 400000～1800000 per pancreas, i.e. about 12150/g pancreas. After purification, the recovery was 350000～1700000 per pancreas, i.e. about 10250/g pancreas, the recovery rate was above 80%, and the purity of the final preparation was above 95%. The insulin secretion in the response to the high concentration glucose (22 mmol/L) stimulation was apparently different on the 1,3,5 day of the cultural islets, which the high level of insulin was three times the low level (5.5 mmol/L) on the 5th day, and the insulin level of the double stimulation under perfusion conditions is apparently higher than low glucose. The result demonstrated that the purified islets were functionally alive. Histological studies also show that the shape of islets are complete, and the β cell was specially stained by the dithizone (DTZ). The Trypan Blue staining had shown the living cell was above 90%. In conclusion, the new method was highly practical and yielded higher concentration of active pancreatic islets.

Isolation, Partial Purification and Characterization of Texas Live Oak (<i>Quercus fusiformis</i>) Lectin

Lectins are carbohydrate-binding proteins with agglutination properties. There is a continuous interest in lectins due to their biological properties that can be exploited for medicinal and therapeutic purposes. The objective of this study was to isolate and characterize lectin activity in Texas Live Oak (Quercus fusiformis). More specifically, the study aimed to determine the lectin’s blood group specificity and pH stability, determine effects of seasonal variation, soil moisture and soil pH on lectin activity. The study also aimed to determine the presence of antifungal activity in Q. fusiformis extracts. Lectin activity was detected and compared via agglutination and protein assays. Protein partial purification was accomplished using diethylaminoethyl ion-exchange chromatography matrix. High Performance Liquid Chromatography (HPLC) was used to assess purity of the lectin. Results showed that Q. fusiformis extracts’ lectin activities are stable at a pH range of 5.2 - 9.2 but with a significant decrease in activity above pH 9.2. The lectin activity was significantly higher when assayed against sheep red blood cells as compared to other blood groups tested. Quercus fusiformis extract is devoid of antifungal activity against Aspergillus niger and Rhizopus stolonifer. The effects of seasonal variation, soil moisture and soil pH do not significantly correlate with lectin activity. Results from HPLC showed presence of three peaks indicating a partial purification of the Q. fusiformis lectin.

ISOLATION AND PURIFICATION OF METHANE IVf ONOOXYGENASE FROM METHYLOMONAS SPECIES GYJ3

A three—component enzyme system that catalyzes in vivo the oxidation of CH_4 to CH_3OH has been purified with high specific activity from an unusual type I methanotroph through the use of stabilizing reagents.

A Review on the Isolation and Purification of D-allulose

D-allulose has very little content in nature,and it needs to be synthesized artificially and meet the purity requirements of industrial grade.The basic physical and chemical properties of D-allulose,its preparation methods and many different ways of isolation and purification were described.In order to achieve the goal of industrial production of D-allulose as soon as possible,the research progress of D-allulose isolation and purification technologies at home and abroad in recent years was classified and discussed,so as to provide useful reference for the practical improvement of D-allulose isolation and purification process technologies.

Purification and Antimicrobial Activity of Antimicrobial Protein from Brown-spotted Grouper, Epinephelus fario

Antimicrobial proteins and peptides had been found from a wide variety of organisms in the last few years These molecules have attracted much research interest because of their biochemical diversity, broad specificity on anti-viral, anti-bacterial, anti-fungi, anti-protozoan parasites, anti-tumoural, and wound-healing effects. Antimicrobial proteins and peptides play key roles in innate immunity. They interact directly with bacteria and kill them. The brown-spotted grouper, Epinephelusfario, is an important marine fish cultured in southem China. Recently, bacteria and virus have caused high mortality in E. fario cultures, but its endogenous antimicrobial peptides and proteins have not been explored. An antimicrobial component was found from the skin homogenate of E. fario. After the skin homogenate was digested with trypsin, its antimicrobial activity was lost, which showed that the antimicrobial component is a protein. The antimicrobial protein （Efap） was purified from the skin homogenate of E. fario by successive ion-exchange and gel filtration chromatography. Efap was demonstrated to be single protein band by SDS-PAGE, with the apparent molecular weight of 41 kD. Efap exhibited antimicrobial activity both for the Gram-positive bacteria, Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis, and for the Gram-negative bacteria, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio fluvialis, Pasteurella multocida, Aeromonas hydrophila, Eschrrichiu coli, and Pseudomonas aeruginosa. Except A. hydrophila, P. aeruginosa, and E. coli （MIC〉20 mol/L）, most of the tested Gram-negative bacteria were sensitive to Efap （MIC〈20 mol/L）. Interestingly, Efap showed potent antimicrobial activity against Gram-positive bacteria S. aureus （MIC 5-10 mol/L） but comparatively weak antimicrobial activity against M. luteus and B. subtilis. The broad antimicrobial activities of Efap suggest that it contributes to the innate host defence of E. fario.

Purification of the Drosophila melanogaster Proteins Inscuteable and Staufen Expressed in Escherichia coli

The proteins Inscuteable and Staufen are key components during asymmetric cell division of neuroblasts for the development of Drosophila melanogaster. Expression and purification of both proteins has been a difficult task for structure-function studies. Based on codon optimization for protein expression in Escherichia coli, we have been able to produce, in soluble form, the C-terminal domains of Inscuteable and Staufen as chimeras with N-terminal maltose binding protein tag that contains a rigid linker between them for feasible crystallization. In addition, using an optimized synthetic gene, corresponding to the amino acid region 250 - 623 of Inscuteable fused to glutathione-S-transferase, low-scale expression experiments showed production of soluble protein. Finally, eukaryotic expression of Inscuteable in the methylothropic yeast Pichia pastoris failed to produce the Drosophila protein at detectable amounts, reinforcing the fact that E. coli still was the microorganism of choice for high-yield protein expression.

Apoptosis and oxidative injury of donor islets during isolation and purification

Objective To observe the changes of islet cell apoptosis and oxidation - antioxidation before transplantation,and to explore pathways of islet protection. Methods Fifteen human pancreases were perfused with Hanks solution containing collagenase,then digested and isolated.