Sepharose Fast Flow Chromatography Research Articles

Microwave assisted extraction, characterization of a polysaccharide from Salvia miltiorrhiza Bunge and its antioxidant effects via ferroptosis-mediated activation of the Nrf2/HO-1 pathway

A microwave-assisted extraction procedure for the crude Salvia miltiorrhiza polysaccharides (SMPs) obtained from Salvia miltiorrhiza Bunge was optimized. Four independent variables were studied: microwave power, extraction time, solvent-to-solid ratio, and concentration of ethanol, with optimal settings of 1200 W, 12 min, 38, and 86 %, respectively. The SMPs were successively purified by DEAE Sepharose Fast Flow and Sephadex G-100 chromatography to produce a novel polysaccharide termed SMP1. The SMP1 was composed of glucose, galactose, and fructose in a molar ratio of 1:1.67:1.12 with an average molecular weight of 6087 Da. Pharmacological studies showed that SMP1 protected from OGD/R-induced ferroptosis and lipid peroxidation by activating Nrf2/HO-1 pathway in PC12 cells. Our research systematically indicated that polysaccharide could inhibit ferroptosis to alleviate oxidative stress injury, which laid the foundation for the future clinical application of Salvia miltiorrhiza polysaccharide.

Structural Characterization and Anti-inflammatory Activity of a Galactorhamnan Polysaccharide From Citrus medica L. var. sarcodactylis.

This study aimed to extract polysaccharides from Citrus medica L. var. sarcodactylis (finger citron fruits) and analyze their structures and potential bioactivities. A new polysaccharide named K-CMLP was isolated and purified by Diethylaminoethylcellulose (DEAE)-Sepharose Fast Flow and DEAE-52 cellulose column chromatography with an average molecular weight of 3.76 × 103 kDa. Monosaccharide composition analysis revealed that K-CLMP consisted of rhamnose, galactose, and glucose, with a molar ratio of 6.75:5.87:1.00. Co-resolved by methylation and two-dimensional nuclear magnetic resonance (NMR), K-CLMP was alternately connected with 1, 2-Rha and 1, 4-Gal to form the backbone, and a small number of glucose residues was connected to O-4 of rhamnose. The results of DPPH⋅ and ABTS+⋅ radical scavenging assays indicated that both crude polysaccharide Citrus medica L. var. polysaccharide (CMLP) and K-CLMP exhibited strong free-radical-scavenging properties in a dose-dependent manner. In addition, K-CMLP significantly inhibited the production of pro-inflammatory cytokines (IL-6 and TNF-α) and reactive oxygen species (ROS) in RAW 264.7 cells treated with LPS. These results provide a basis for further use as one of the potential functions of food or natural medicine.

Extraction, purification and characterization of sulphated polysaccharide from Bellamya quadrata and its stabilization roles on atherosclerotic plaque

The aim of this paper was to investigate the extraction, purification and characterization of sulphated polysaccharide (BQPS) from Bellamya quadrata and its stabilization roles on atherosclerotic plaque. Firstly, crude polysaccharide (CBQP) from Bellamya quadrata was extracted by protease enzyme assisted extraction. Moreover, its optimal parameters were obtained by the response surface method as follows: the ratio of water to raw material of 24:1, enzyme dosage of 285 U/g, enzymolysis pH value of 4.7 and temperature of 67 °C. Secondly, CBQP was further purified to obtain the target polysaccharide BQPS by Q Sepharose Fast Flow and Sephacryl S-400 gel column chromatography. Then, the characterization of BQPS revealed that it possessed a total polysaccharide content of 91.88 ± 1.23%, sulfuric acid group content of 9.12 ± 1.59% and molecular weight of 91.1 kDa. BQPS was only consisted of glucose without any proteins. Finally, BQPS was confirmed to have a significant stabilizing effect on atherosclerotic plaque and its mechanism was related to the selective promotion of autophagy with the precisely right strength.

Purification of R-phycoerythrin from a marine macroalga Gracilaria gracilis by anion-exchange chromatography

Gracilaria gracilis, a red macroalga, represents an exploited important biomass. One of the potential uses of this red seaweed is the production of valuable molecules such as R-phycoerythrin. This compound has recently found applications in food and cosmetic industries as a natural colorant, in conducting medical diagnosis and in biology. However, after R-phycoerythrin extraction the protein extract has to be concentrated and pre-purified, which is commonly expensive and requires many steps. In this paper, R-phycoerythrin from the crude extract obtained by phosphate buffer 20 mM, pH 7.1, was purified by one-step chromatographic method. Native R-phycoerythrin was achieved with a high purity index (A565/A280 ratio) of 3.25 (3.2 = standard R-phycoerythrin purity) after purification on DEAE Sepharose fast flow chromatography with the obtained fraction at 200 mM NaCl. This fraction presented the absorption and emission spectra of R-phycoerythrin with major absorbance at 498, 540, and 565 nm. Native form of this molecule was presented about 260 kDa. Besides, the purified pigment R-phycoerythrin included four subunits: α subunit (18 kDa), β subunit (21 kDa), γ subunit (29 kDa), and γ’ subunit (27 kDa) by gel electrophoresis.

Chemistry and immunostimulatory activity of a polysaccharide from Undaria pinnatifida

An immunologically active polysaccharide named as UPP-2 (1035.52 kDa) was isolated from Undaria pinnatifida using traditional water extraction followed by DEAE Sepharose fast flow chromatography. UPP-2 was proven to be a low sulfated polysaccharide with relatively abundant uronic acid (13.08 ± 0.67%). UPP-2 mainly consisted of xylose (64.55%), glucose (23.81%), arabinose (5.90%) and mannose (4.26%), and its main glycosidic linkage types included →2)-α-D-Xylp-(1→, →4)-α-D-Glcp-(1→, α-D-Xylp-(1→ and →2,4)-β-D-Xylp-(1 → . Results indicated that UPP-2 significantly promoted the proliferation and pinocytic capacity of RAW264.7 cells, and upregulated the mRNA expressions of iNOS, TNF-α, IL-6 and IL-1β at 100–600 μg/mL with a maximum of 195, 42, 768 and 539 times of those of the negative control, respectively. Moreover, UPP-2 significantly increased the secretions of nitric oxide, TNF-α and IL-6 at 100–600 μg/mL (8.0, 73.1 and 188.7 times compared to those of the negative control, respectively), as well as promoted the production of IL-1β obviously at 600 μg/mL. Overall, UPP-2 could be served as a potential dietary supplement or functional food based on its immunostimulatory activity.

Characterization of polysaccharide from Helicteres angustifolia L. and its immunomodulatory activities on macrophages RAW264.7

Helicteres angustifolia L. (H. angustifolia) has been widely used as a remedy against various types of illness relating to immune response, such as inflammations and fever. In order to characterize the structure and identify the immunomodulatory activity of polysaccharide from H. angustifolia, a polysaccharide fraction (SPF3-1) was purified from H. angustifolia by using DEAE Sepharose Fast Flow and Sephacry S-400 chromatography, successively. Physicochemical analysis demonstrated that SPF3-1 is an acidic heteropolysaccharide with a molecular weight of about 13.36 kDa; in vitro immunomodulatory assay reflects that SPF3-1 could significantly (p < 0.05) enhance the proliferation of macrophages, stimulate the macrophages phagocytic capacity, as well as induce NO and immunomodulatory cytokines generation. All the results suggest that SPF3-1 from H. angustifolia possesses potent immunomodulatory activity and could be further developed as new products for medicines or functional foods.

Expression, purification and biological activity of monomeric insulin precursors from methylotrophic yeasts

The methylotrophic yeasts Pichia pastoris and Hansenula polymorpha have been used for the production of recombinant monomeric insulin precursor (MIP). Recombinant plasmids with one, two and four cassettes of the MIP gene have been successfully constructed in the pPICZαA expression vector to study the effects of gene copy number on MIP production. The MIP protein can be detected by dot-blot analysis from the culture broth of P. pastoris KM71H 24 h after placement in MMH induction medium. The secretion levels of MIP protein in culture broth at 72 h after induction indicated that P. pastoris KM71H with one cassette of the MIP gene had highest MIP protein levels (4.19 ± 0.96 mg L−1). The transcription levels of the MIP gene increased proportionately with copy number. However, the amount of secreted MIP protein showed no correlation. The MIP molecular mass was 5756.951 Da, as confirmed by typical MALDI-TOF mass spectrometry. The MIP protein in culture broth was purified by two steps purification including SP Sepharose Fast Flow chromatography followed by ultrafiltration (10 kDa MW cutoff). The percentage of MIP recovery after the two-step purification was 70%, with a single band in a native-PAGE. The biological activity of tryptic hydrolyzed MIP was determined via the expression of the glucose transporter 4 gene (GLUT4) in H9c2 (2-1) cell line by RT-qPCR, and the results demonstrated that the MIP protein can induce glucose uptake and upregulation of GLUT4 mRNA transcription at 3 h and that this activity was related to Humalog® insulin.

Extraction Optimization, Purification, Antioxidant Activity, and Preliminary Structural Characterization of Crude Polysaccharide from an Arctic Chlorella sp.

The arctic strain of Chlorella sp. (Chlorella-Arc) exists in the coldest and driest arctic ecosystems, and it is a new resource of active polysaccharides. The extraction of crude polysaccharide from Chlorella-Arc was optimized using the response surface methodology. A crude polysaccharide yield of approximately 9.62 ± 0.11% dry weight was obtained under these optimized conditions. Three fractions (P-I, P-II, and P-III) were present after purification by 2-diethylaminoethanol Sepharose Fast Flow and Sephadex G-100 chromatography. The P-IIa fraction demonstrated significant antioxidant activities. Moreover, P-IIa was an α- and β-type heteropolysaccharide with a pyran group and contained variable amounts of rhamnose, arabinose, glucose, and galactose based on fourier-transform infrared spectroscopy, high-performance liquid chromatography, and 1H and 13C nuclear magnetic resonance imaging. Production of high amounts of polysaccharide may allow further exploration of the microalgae Chlorella-Arc as a natural antioxidant.

Expression of adenylate kinase fused MEK1R4F in Escherichia coli and its application in ERK phosphorylation.

To construct a highly expressed and active MEK1R4F (a constitutively active form of mitogen-activated protein kinase kinase 1) by fusion of soluble adenylate kinase (Adk) tag, resulting in Adk-MEK1R4F protein suitable for preparation of phosphorylated ERK. We fused the Adk to the N-terminus of MEK1R4F through overlapping PCR. The expression of Adk-MEK1R4F fusion protein increased ~10-fold in Escherichia coli, and was purified to 95% via two purification steps including Ni-NTA and Q Sepharose fast flow (QFF) chromatography. The purified Adk-MEK1R4F protein was functional for ERK phosphorylation and could use ADP in addition to ATP. The Adk-MEK1R4F had higher catalytic activity than regular MEK1R4F both in vitro and in cell-free extracts system. Adenylate kinase was used as a soluble tag to facilitate MEK1R4F protein expression and its application in large-scale phosphorylated ERK1/2 preparation and purification.

Increased production of human fibroblast growth factor 17 in Escherichia coli and proliferative activity in NIH3T3 cells.

Fibroblast growth factor 17 (FGF17) is a novel member of the FGFs family, which is essential for cell development, tissue repair, tumor growth and invasion. The aim of the current study was to obtain a high expression level of recombinant human FGF17 (rhFGF17), including soluble proteins and inclusion bodies. An optimized rhFGF17 cDNA sequence was cloned into a pET3a vector, then the pET3a‑hFGF17 vector was transformed into BL21(DE3)pLysS Escherichiacoli cells. Expression was induced by optimizing the conditions using isopropyl β‑D‑1‑thiogalactopyranoside (IPTG) and it was confirmed that a 24‑h exposure to 0.8mM IPTG at 16˚C provided the optimal condition for soluble hFGF17. Furthermore, for the inclusion bodies, the optimal condition was a 4‑h exposure to 0.4mM IPTG at 37˚C. Two forms of rhFGF17 protein were purified by heparin affinity and SP Sepharose Fast Flow chromatography. MTT assays demonstrated that the purified rhFGF17 exerted an important effect on the proliferative activity of NIH3T3 cells, although there was no significant difference when compared with standard rhFGF17. Thus, an optimal and economic expression system was created in the present study for rhFGF17 in E. coli. This expression strategy enables the preparation of sufficient and highly bioactive rhFGF17 for further investigation of underlying mechanisms.

Structure Studies of the Extracellular Polysaccharide from Trichoderma sp. KK19L1 and Its Antitumor Effect via Cell Cycle Arrest and Apoptosis.

An extracellular polysaccharide TP1A was purified from the fermented broth of Trichoderma sp. KK19L1 by combination of Q Sepharose fast flow and Sephacryl S-300 chromatography. TP1A was composed of Man, Gal, and Glc in a molar ratio of about 3.0:5.1:8.1. The molar mass of TP1A was about 40.0kDa. Methylation and NMR analysis indicated that the probable structure of TP1A was [→4,6)-α-D-Glcp(1→6)-β-D-Galf(1→6)-β-D-Galf(1→2,6)-β-D-Galf(1→2,6)-β-D-Galf(1→2,6)-β-D-Galf(1→2,6)-α-D-Manp(1→2,6)-α-D-Manp(1→] with [α-D-Glcp(1→] and [α-D-Manp(1→6)-α-D-Glcp(1→6)-α-D-Glcp(1→] as branches. The antitumor study showed that TP1A was able to inhibit the cell viability of HeLa and MCF-7 cells. TP1A could arrest HeLa cells in G2/M phase and induce HeLa cell apoptosis. These findings suggest that fungal polysaccharides could be a potential source for antitumor agents.

Purification and Molecular Characterization of Polysaccharides from Gentianae Radix et Rhizoma by DEAE Sepharose Fast Flow Chromatography

Purification and Molecular Characterization of Polysaccharides from Gentianae Radix et Rhizoma by DEAE Sepharose Fast Flow Chromatography

Structural Characterization and Immunological Activities of a Novel Water-Soluble Polysaccharide from the Fruiting Bodies of Culinary-Medicinal Winter Mushroom, Flammulina velutipes (Agaricomycetes).

A water-soluble polysaccharide, designated FVPA2, was isolated from the fruiting bodies of Flammulina velutipes using DEAE Sepharose Fast Flow and gel-permeation chromatography. Its structure was elucidated by monosaccharide composition and methylation analysis, ultraviolet, Fourier transform infrared spectrometry, and nuclear magnetic resonance spectroscopy. Results showed that FVPA2 was a homogeneous heteropolysaccharide containing galactose, fucose, and mannose in a molar ratio of 5:1:1. High-performance liquid chromatography indicated its molecular weight as 3.4 × 104 Da. FVPA2 also has a repeating unit. In vitro immunomodulatory studies showed that Raw264.7 cells were stimulated to secret nitric oxide upon administration of 200-500 µg/mL FVPA2. FVPA2 also stimulated the proliferation of mouse spleen lymphocytes and B lymphocytes.

High-level expression and characterization of a thermostable xylanase mutant from Trichoderma reesei in Pichia pastoris

A gene encoding xylanase 2 mutant from Trichoderma reesei (T2C/T28C, named mxyn2) was cloned into the Pichia pastoris X33 strain using the vector pPICZαA. Recombinant Mxyn2p was functionally expressed in P. pastoris X33 and secreted into the supernatant. Real time qPCR demonstrated that an increase in gene copy number correlated with higher levels of expression. Supernatant from methanol induced cells was concentrated by ultrafiltration with a 10kDa cut off membrane, and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. Recombinant Mxyn2p protein had the highest activity at 75°C, while recombinant protein encoded by the “wild type” xylanase gene xyn2, also expressed in Pichia, was 20°C lower. The Mxyn2p enzyme retained more than 70% of its activity after incubation at 80°C for 10min. The effects of the optimal pH and temperature for higher expression levels in P. pastoris were also determined, 6.0 and 22°C, respectively. The maximum xylanase activity of Mxyn2p was 13,000nkat/mg (9.88g/l) in fed-batch cultivation after 168h induction with methanol in a 50l bioreactor.

A novel and efficient method for the isolation and purification of polysaccharides from lily bulbs by Saccharomyces cerevisiae fermentation

A water-soluble polysaccharide from lily bulbs was isolated and purified by Saccharomyces cerevisiae fermentation. Proteins present in lily bulb extract were removed by extracellular proteases secreted by S. cerevisiae during fermentation. This novel method differs from traditional protein removal methods. A suitable yeast strain was selected. Culture conditions were optimized. Response surface methodology (RSM) was utilized to evaluate the effects of variables on the lily polysaccharide (LP) yield and the protein removal ratio (PRR). The results of applying RSM revealed that the optimum fermentation conditions were 87.5gL−1 lily bulb powder, pH 5.6, and temperature 27.9°C. When lily bulb extract was cultured with S. cerevisae under optimum conditions, the LP yield and the PRR were 6.56% and 91.46%, respectively. These values are in close agreement with the value predicted by the model. The resulting LP curding was further purified by DEAE Sepharose Fast Flow chromatography after isolation by alcohol precipitation post-fermentation. DEAE chromatography resulted in a fraction, LP-1 (yield: 4.46%) with a molecular weight of 65.0kDa. LP-1 consisted of glucose and mannose in a molar ratio of 1:1.2.

Purification, preliminary characterization and in vitro immunomodulatory activity of tiger lily polysaccharide

A water-soluble polysaccharide (LLPS) from tiger lily was extracted by ultrasonic wave-assisted extraction. The LLPS, which was isolated by alcohol precipitation, was further purified by DEAE Sepharose Fast Flow and Sephadex G-100 chromatography, which resulted in LLPS fractions in LLPS-1, LLPS-2 and LLPS-3, with molecular weights of 350.5, 403.3 and 146.2kDa, respectively. LLPS-1 and LLPS-2 primarily consisted of glucose and mannose in a molar ratio of nearly 1:2 and 1:1, respectively. In contrast, LLPS-3 was primarily composed of arabinose, galactose, glucose and mannose in a molar ratio of nearly 2:2:2:1. LLPS fractions could stimulate the proliferation of macrophages. The in vitro immunomodulatory activity of the fractions was evaluated. The results showed that treatment with 25–400μg/mL of LLPS fractions could increase phagocytic activity and nitric oxide production of macrophages in a dose-dependent manner.

High-Level Expression of Pro-Form Lipase from Rhizopus oryzae in Pichia pastoris and Its Purification and Characterization

A gene encoding Rhizopus oryzae lipase containing prosequence (ProROL) was cloned into the pPICZαA and electrotransformed into the Pichia pastoris X-33 strain. The lipase was functionally expressed and secreted in Pichia pastoris with a molecular weight of 35 kDa. The maximum lipase activity of recombinant lipase (rProROL) was 21,000 U/mL, which was obtained in a fed-batch cultivation after 168 h induction with methanol in a 50-L bioreactor. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut off membrane and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. The optimum pH and temperature of the rProROL were pH 9.0 and 40 °C, respectively. The lipase was stable from pH 4.0 to 9.0 and from 25 to 55 °C. The enzyme activity was enhanced by Ca2+ and inhibited by Hg2+ and Ag+. The lipase showed high activity toward triglyceride-Tripalmitin (C16:0) and triglyceride-Trilaurin (C12:0).

Expression, purification and characterization of a recombinant Tat47–57-Oct4 fusion protein in Pichia pastoris

The transcription factor, Oct-4, is involved in the self-renewal of undifferentiated embryonic stem cells, and is also significant in the reprogramming process and in the development of tumors. In the present study, the fusion protein, Tat47‑57-Oct4, was secreted by the signal peptide of human serum albumin in Pichia pastoris under the control of alcohol oxidase promoter 1. The yield of recombinant Tat47‑57-Oct4 fusion protein was ~210 mg/l. Following pilot‑scale fermentation, Tat47‑57-Oct4 was purified by ammonium sulfate precipitation, Vivaflow 200 ultrafiltration and SP Sepharose fast flow chromatography in order to obtain 95.6% purity. Immunofluorescence analysis validated the ability of Tat47‑57-Oct4 to cross the cell membrane. The results demonstrated that the experimental procedure developed in the present study could produce large quantities of active Tat47‑57-Oct4 fusion protein from P. pastoris.

Purification and Characterization of Polyphenol Oxidase From Agaricus bisporus

A polyphenol oxidase was isolated from Agaricus bisporus using ammonium sulfate precipitation, combined with DEAE–Sepharose Fast Flow chromatography and Phenyl Sepharose CL-4B chromatography. The molecular weight of the purified polyphenol oxidase was determined to be about 43 kDa with N-terminal sequence Ala-Thr-Asn-Ser-Gly-Thr-Leu-Ile-Ile-Phe by Edman degradation. The optimum temperature and pH for the polyphenol oxidase activity was 20°C and a pH of 6.5–7.0. Moreover, it was stable at 30 and 40°C over a 60 min pre-incubation time period. The Km and Vmax values of this polyphenol oxidase for catechol were 0.67 mM and 3333 U/ml min−1, respectively. In addition, following the cross linking with polyphenol oxidase, the emulsifying activity index, foam capacity, and foam stability of the whey proteins were evaluated to be modified. This work was useful to better understand polyphenol oxidase from A. bisporus, and may lead to its practical use in the future.

Porcine circovirus (PCV) removal by Q sepharose fast flow chromatography.

The recently discovered contamination of oral rotavirus vaccines led to exposure of millions of infants to porcine circovirus (PCV). PCV was not detected by conventional virus screening tests. Regulatory agencies expect exclusion of adventitious viruses from biological products. Therefore, methods for inactivation/removal of viruses have to be implemented as an additional safety barrier whenever feasible. However, inactivation or removal of PCV is difficult. PCV is highly resistant to widely used physicochemical inactivation procedures. Circoviruses such as PCV are the smallest viruses known and are not expected to be effectively removed by currently-used virus filters due to the small size of the circovirus particles. Anion exchange chromatography such as Q Sepharose® Fast Flow (QSFF) has been shown to effectively remove a range of viruses including parvoviruses. In this study, we investigated PCV1 removal by virus filtration and by QSFF chromatography. As expected, PCV1 could not be effectively removed by virus filtration. However, PCV1 could be effectively removed by QSFF as used during the purification of monoclonal antibodies (mAbs) and a log10 reduction value (LRV) of 4.12 was obtained. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1464–1471, 2013