胸腺素a1与复合a干扰素融合蛋白的表达及其生物学活性

实验旨在获得具有双重生物学活性的重组胸腺素a1(Thymosin alpha1, TM-a1)与复合a干扰素(IFNa-con)融合蛋白。选择大肠杆菌偏爱的密码子, 将合成的TM-a1与IFNa-con编码序列构成的融和基因克隆至大肠杆菌表达载体pET-22b(+)、在宿主菌BL21(DE3)-Codon plus-RP-X中成功表达了可溶性融合蛋白(TM-a1-IFN-con)。表达量占总蛋白的20%以上。通过硫酸铵沉淀、疏水层析、阴离子交换层析、阳离子交换层析、分子筛层析后, 产品纯度达到96%以上。采用细胞病变抑制法测定融合蛋白的抗病毒活性, 采用细胞增殖实验检测融合蛋白对小鼠脾淋巴细胞增殖的影响。结果表明, 融合蛋白的抗病毒活性优于市售的IFNa1b和IFNa2a。对小鼠脾淋巴细胞增殖的影响与市售的合成胸腺素a1相同。已有研究证实, 该融合蛋白具有良好的体外抗HBV作用, 其体外抗HBV活性比联合应用TM-a1和干扰素a强, 且细胞毒性明显低于联合应用TM-a1和干扰素a。以上结果表明, 通过大肠杆菌表达的可溶性融合蛋白(TM-a1- IFN-con), 既具有良好的干扰素a抗病毒作用, 也具有胸腺素a1促淋巴细胞增殖作用。     

一种新的蛇毒凝血酶原激活物的分离纯化及特征

为获得矛头蝮蛇（Bothrops atrox）蛇毒凝血酶原激活物并研究其基本性质,采用SP Sepharose Fast Flow, DEAE Sepharose Fast Flow 和SP Sepharose High Performance等层析方法从巴西矛头蝮蛇蛇毒中分离纯化得到1种单一组分的凝血酶原激活物（prothrombin activator,FⅡA）.还原性SDS-PAGE结果显示,其分子质量约为72 kD,等电点为6.67. HPSEC显示纯度大于95%.该酶是1种N连接的糖蛋白,N末端氨基酸序列为ALVLIAFAQYLQQCP,获得登录号为：B3A0N1其活性可被EDTA-Na₂ 抑制,PMSF对其活性无影响,对凝血酶原的激活过程无需Ca2+、FⅤa、磷脂的参与,为P-Ⅰ金属蛋白酶,对凝血酶原的激活方式与FⅩa相似.本研究纯化与鉴定的新凝血酶原激活物为其药学研究及临床应用提供参考.     

Characteristics of the adsorption of immunoglobulin M onto Q Sepharose® Fast Flow ion-exchangers

Measurement of adsorption breakthrough curves in packed beds has shown that the amounts and rates of uptake of immunoglobulin M (IgM) onto the commonly used anionic ion-exchanger Q Sepharose Fast Flow(based on 6% agarose) are severely limited as a result of the large molecular size of this adsorbate(RMM 950000). A similar ion-exchanger based on a more porous 4% agarose, Q Sepharose 4 Fast Flow was evaluated as an alternative adsorbent for the purification of IgM. Equilibrium adsorption isotherms and the effective diffusivities of IgM within these two adsorbents were measured. Q-Sepharose 4 Fast Flow was found to have a maximum capacity for IgM 2.5 times greater than that of Q Sepharose 6 Fast Flow and the effective diffusivity of IgM was found to be between 6 and 7 times greater than with the latter material. Comparison of the breakthrough curves obtained for these adsorbents at a variety of flow velocities confirm that Q Sepharose 4 Fast Flow is a superior adsorbent for the capture and purification of large proteins.This revised version was published online in October 2005 with corrections to the Cover Date.     

A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, a Chinese medicinal herb

The immunopotentiating effect of the roots of Astragalus membranaceus, a medicinal herb, has been associated with its polysaccharide fractions (Astragalus polysaccharides, APS). We herein demonstrate that APS activates mouse B cells and macrophages, but not T cells, in terms of proliferation or cytokine production. Fluorescence-labeled APS (fl-APS) was able to selectively stain murine B cells, macrophages and a also human tumor cell line, THP-1, as determined in flow cytometric analysis and confocal laser scanning microscopy. The specific binding of APS to B cells and macrophages was competitively inhibited by bacterial lipopolysaccharides. Rabbit-anti-mouse immunoglobulin (Ig) antibody was able to inhibit APS-induced proliferation of, and APS binding to, mouse B cells. Additionally, APS effectively stimulated the proliferation of splenic B cells from C3H/HeJ mice that have a mutated TLR4 molecule incapable of signal transduction. These results indicate that APS activates B cells via membrane Ig in a TLR4-independent manner. Interestingly, macrophages from C3H/HeJ mice were unable to respond to APS stimulation, suggesting a positive involvement of the TLR4 molecule in APS-mediated macrophage activation. Monoclonal Ab against mouse TLR4 partially inhibited APS binding with macrophages, implying direct interaction between APS and TLR4 on cell surface. These results may have important implications for our understanding on the molecular mechanisms of immunopotentiating polysaccharides from medicinal herbs.     

A new procedure for fast isolation and purification of plastocyanin from the cyanobacterium Anabaena variabilis

Methods are described for growing the cyanobacterium A. variabilis and for the isolation and purification of plastocyanin from the grown culture. Cell paste which had been stored at –35°C was suspended in 1 mM MES buffer, pH 6.5 and centrifuged. The supernatant was diluted to a conductivity of 0.12 mS, [Fe(CN)₆]^3- added to a concentration of 0.5 mM and the solution loaded on a S Sepharose Fast Flow column. After elution and ultrafiltration, the plastocyanin containing fractions were reloaded on a S Sepharose Fast Flow column for final purification. A typical yield in three days from cells harvested from 3×20 l of medium was 32 mg plastocyanin with a minimum absorbance ratio A₂₇₈/A₅₉₇=1.14. This procedure is faster and the yield higher than for previous procedures.Abbreviations MES 2(N-morpholino)ethanesulfonic acid - PC plastocyanin     

Isolation and characterization of an antifungal protein from Bacillus licheniformis HS10

Bacillus licheniformis HS10 is a good biocontrol agent against Pseudoperonospora cubensis which caused cucumber downy disease. To identify and characterize the antifungal proteins produced by B.licheniformis HS10, the proteins from HS10 were isolated by using 30–60% ammonium sulfate precipitation, and purified with column chromatography on DEAE Sepharose Fast Flow, RESOURCE Q and Sephadex G-75. And the SDS–PAGE and MALDI-TOF/TOF-MS analysis results demonstrated that the antifungal protein was a monomer with molecular weight of about 55 kDa, identified as carboxypeptidase. Our experiments also showed that the antifungal protein from B. licheniformis HS10 had significantly inhibition on eight different kinds of plant pathogenic fungi, and it was stable with good biological activity at as high as 100 °C for 30 min and in pH value ranged from 6 to 10. The biological activity was negatively affected by protease K and 10 mM metal cations except Ca²⁺.     

Isolation and antioxidant activities of polysaccharides extracted from the shoots of Phyllostachys edulis (Carr.)

Polysaccharides extracted from Phyllostachys edulis (Carr.) are a group of hetero polysaccharides, and their antioxidant activities were investigated employing various established in vitro systems. Available data obtained with in vitro models suggested that among the three samples, B1 (extraction with water) showed significant inhibitory effects on superoxide radical and hydroxyl radical; its reducing power was also the strongest among the three samples. These results clearly establish the possibility that polysaccharides extracted from P. edulis could be effectively employed as ingredient in health or functional food, to alleviate oxidative stress. However, comprehensive studies need to be conducted in experimental animal models.     

Immunomodulation and antitumor activities of different-molecular-weight polysaccharides from Porphyridium cruentum

The extracellular polysaccharides (EPSs) isolated from Porphyridium cruentum were degraded by hermetical-microwave and H₂O₂ under ultrasonic waves. Six products were obtained with molecular weights of 6.53, 256, 606, 802.6, 903.3 and 1002 kDa. The antitumor and immunomodulatory activities of different-molecular-weight (MW) polysaccharides were evaluated by the S180-tumor-bearing mouse model in vivo and peritoneal macrophage activation in vitro. The degraded EPSs all showed clear immunomodulation to different extents. The MW of the EPSs had a notable effect on their activity. The 6.53-kDa fragment had the strongest immunoenhancing activity. Different doses of EPS all inhibited the growth of the implanted S180 tumor. The tumor inhibition index at high, middle and low doses was 53.3%, 47.5% and 40.5%, respectively. In addition, three different concentrations of EPS significantly increased lymphocyte proliferation, which indicated the unique mechanism of the antitumor effect of EPS.     

Purification of histidine-tagged nucleocapsid protein of Nipah virus using immobilized metal affinity chromatography

Nucleocapsid (N) protein of Nipah virus (NiV) is a potential serological marker used in the diagnosis of NiV infections. In this study, a rapid and efficient purification system, HisTrap? 6 Fast Flow packed bed column was applied to purify recombinant histidine-tagged N protein of NiV from clarified feedstock. The optimizations of binding and elution conditions of N protein of NiV onto and from Nickel Sepharose? 6 Fast Flow were investigated. The optimal binding was achieved at pH 7.5, superficial velocity of 1.25 cm/min. The bound N protein was successfully recovered by a stepwise elution with different concentration of imidazole (50, 150, 300 and 500 mM). The N protein of NiV was captured and eluted from an inlet N protein concentration of 0.4 mg/ml in a scale-up immobilized metal affinity chromatography (IMAC) packed bed column of Nickel Sepharose? 6 Fast Flow with the optimized condition obtained from the method scouting. The purification of histidine-tagged N protein using IMAC packed bed column has resulted a 68.3% yield and a purification factor of 7.94.     

Purification of Bovine α-Lactalbumin by Immobilized Metal Ion Affinity Chromatography

ABSTRACT

The milk protein α-lactalbumin was isolated from bovine whey protein concentrate solution by immobilized metal ion affinity chromatography (MAC) using Cu(II)-Chelating Sepharose Fast Flow. Stepwise pH (5.5–3.8) changes in sodium acetate buffer were used to elute the protein selectively, at which time it was concentrated and reapplied to an uncharged Chelating Sepharose Fast Flow column to remove the contaminating Cu(II) ions. A purity of 90% and recovery of 80% was achieved. The described method appears to be suitable for isolation of a-lactalbumin in a form adequate for milk formula engineering.     

Characterization of polysaccharides from Hypnea spinella (Gigartinales) and Halopithys incurva (Ceramiales) and their effect on RAW 264.7 macrophage activity

Red algae have been reported to be an important source of polysaccharides with potential immunomodulatory properties. The objective of this study was to characterize the polysaccharides from Halopithys incurva and Hypnea spinella and to evaluate their effect on the synthesis of cytokines by murine cell line RAW 264.7 macrophages. Polysaccharides were obtained by N-cetylpyridinium bromide precipitation and characterized by Fourier transform-infrared spectroscopy. Their effect on the activity of RAW 264.7 macrophages was examined by quantification of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and nitric oxide (NO) production using enzyme-linked immunosorbent assays. The activation of the cytokine IL-6 and NO increased linearly as the concentration of polysaccharides from H. incurva and Hy. spinella increased. In general, the activation of IL-6 and NO was tenfold greater when macrophages were exposed to polysaccharides from H. incurva than when exposed to polysaccharides from Hy. spinella. In contrast, TNF-α concentration did not increase when macrophages were exposed to increasing polysaccharide levels. These results indicate that polysaccharides are strong cytokine IL-6 inducers.     

In vitro antioxidant activities of sulfated polysaccharide fractions extracted from Corallina officinalis

Sulfated polysaccharides (F1, F2) from seaweed Corallina officinalis were isolated through anion-exchange column chromatography. Their chemical characteristics were determined by GC, HPLC, FT-IR and UV spectra. F1 and F2 contained only two monosaccharides, namely galactose and xylose. The antioxidant activities of F1, F2 and the de-sulfated polysaccharides (DF-1, DF-2) in vitro were investigated, including hydroxyl radicals scavenging effect, superoxide radical scavenging capacity, DPPH radical activity and reducing power. As expected, antioxidant assay showed that the two sulfated polysaccharide fractions (F1, F2) possessed considerable antioxidant properties and had more excellent abilities than de-sulfated polysaccharides (DF-1, DF-2).     

变色栓菌锰过氧化物酶同工酶的纯化及其性质研究

变色栓菌(Trametes versicolor)胞外产酶培养液经硫酸铵沉淀、DEAE-cellulose DE52离子交换柱层析后,获得两个活性组分D1和D2,其中活性组分D2经Phenyl SepharoseTM6Fast Flow疏水层析后,所得样品MnP1经SDS-PAGE检测已达到电泳纯。活性组分D1经Phenyl SepharoseTM6Fast Flow疏水层析、Sephacryl S-200HR凝胶过滤层析后,所得样品MnP2经SDS-PAGE检测已达到电泳纯。两种同工酶MnP1及MnP2,各自的比活力为579.09、425.00U/mg;纯化倍数为17.51、12.85;活力回收率为6.17%、2.47%。由SDS-PAGE法测得MnP1及MnP2的表观分子量分别为46.3kD、43.0kD。两种同工酶催化DMP(2,6-二甲氧基酚)氧化反应的最适pH值及最适反应温度有所不同,最适pH值分别为pH5.8、pH6.2,最适反应温度分别为60℃、65℃。在45℃以下,pH4.0~7.0之间,MnP1及MnP2的稳定性好。DMP为最佳酶促反应底物,以DMP为底物的Km分别为13.43μmol/L、12.45μmol/L。在无Mn2 存在的条件下,酶促反应几乎不发生。EDTA在较高浓度时抑制酶的活性,DTT在所试浓度下都完全抑制酶的活性。     

Hydrophobic interaction chromatography selectivity changes among three stable proteins: conformation does not play a major role

Interesting retention and selectivity changes have been noted for a number of proteins in hydrophobic interaction chromatography (HIC). In this study, we investigated the degree to which conformational changes may be responsible for selectivity changes of stable proteins. Hydrogen-deuterium isotope exchange detected by mass spectrometry was used to investigate changes in solvent accessibility during adsorption on HIC media. Lysozyme was determined to exhibit EX2 hydrogen exchange kinetics both in solution and adsorbed to Butyl Sepharose 4 Fast Flow and Phenyl Sepharose 6 Fast Flow high sub surfaces. A small, but significant, increase in solvent accessibility was observed upon adsorption. Similar approaches were used to analyze solvent accessibility of three stable proteins with melting temperatures above 50 degrees C exhibiting significant selectivity changes on Butyl Sepharose and Toyopearl Butyl 650M. While all three proteins (lysozyme, chymotrypsinogen A, and ovalbumin) exhibited enhanced exchange while adsorbed, no differences in solvent accessibility on the different adsorbents were observed. More detailed studies of lysozyme showed no significant changes in labeling prior or during elution. These results demonstrate that HIC surfaces examined here do not dramatically alter the structure of these stable proteins and that differences in conformation are not responsible for the selectivity changes observed. Thus, other factors such as different preferred binding orientations or variations between the media pore structure, size, and/or surface chemistry must be responsible.     

Adsorption of BSA on strongly basic chitosan: Equilibria

Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a new adsorbent, a strongly basic crosslinked chitosan (Chitopearl 2503), which is hard and is not compressed by pressure in a column, have been presented and compared with diethylaminoethyl (DEAE) Sepharose Fast Flow (hard gel). In Chitopearl 2503, when only buffer existed in the BSA solution, the isotherm was not affected by the initial concentration of BSA but it was affected by pH considerably. The isotherm was favorable when pH >/= pl ( congruent with 4.8). When NaCl existed in the BSA solution, the amount of BSA absorbed on the resin decreased with increasing concentration of NaCl. When the concentration of NaCl was 200 mol/m(3), the resin did not adsorb BSA at all. The equilibrium data were correlated by the Langmuir equation reasonably well. The BSA may be adsorbed mainly by electrostatic attraction between negatively charged BSA and positively charged quanternary ammonium groups at pH > pl and by protonation reaction of the primary ammonium groups by weak acid groups of BSA at pH = pl. These are confirmed by measuring the amount of inorganic ion exchanged for BSA. In DEAE Sepharose Fast Flow, the isotherm was favorable when pH > pl but unfavorable ar pH = pl. The saturation capacity of BSA on Chitopearl 2503 is about 1.3 to 2.2 times larger than that on DEAE Sepharose Fast Flow. (c) 1994 John Wiley & Sons, Inc.     

Immunologically active polysaccharides of eupatorium cannabinum and eupatorium perfoliatum

From the alkaline aqueous extract of the herbal part of Eupatorium cannabinum and Eupatorium perfoliatum two homogeneous polysaccharides (PI and PII) have been isolated by ethanol precipitation and fractionation on DEAE Sepharose CL-6B and Sephacryl S-400 columns. The structural elucidation was achieved mainly by permethylation, periodate oxidation, basic degradation, methanolysis and reduction experiments, and ¹³C NMR spectrometry. Both polysaocharides, identified as 4-O-methylglucuronoxylans, differ in their M,s (> 500 000 and 40 000) only. The polysaccharides show a phagocytosis enhancing effect as determined in three immunological test systems (carbon clearance, granulocyte- and chemiluminescence test).     

Bacisubin, an antifungal protein with ribonuclease and hemagglutinating activities from Bacillus subtilis strain B-916

An antifungal protein, with a molecular mass of 41.9 kDa, and designated as bacisubin, was isolated from a culture of Bacillus subtilis strain B-916. The isolation procedure consisted of ion exchange chromatography on DEAE-Sepharose Fast Flow, and fast protein liquid chromatography on Phenyl Sepharose 6 Fast Flow and hydroxyapatite columns. The protein was adsorbed on all three chromatographic media. Bacisubin exhibited inhibitory activity on mycelial growth in Magnaporthe grisease, Sclerotinia sclerotiorum, Rhizoctonia solani, Alternaria oleracea, A. brassicae and Botrytis cinerea. The IC50 values of its antifungal activity toward the last four fungal species were 4.01 microM, 0.087 microM, 0.055 microM and 2.74 microM, respectively. Bacisubin demonstrated neither protease activity, nor protease inhibitory activity. However, it manifested ribonuclease and hemagglutinating activities.     

Ultrasound complex enzymes assisted extraction and biochemical activities of polysaccharides from Epimedium leaves

Ultrasound complex enzymes (UCE) were used to extract crude polysaccharides (CEP) from Epimedium leaves. The response surface methodology (RSM) based on Box–Behnken Design (BBD) was employed to further optimize the extraction conditions. The optimum conditions were: complex enzyme dose of 50, 250, 200, 100 U/g (papain, pectase, cellulase and α-amylase), extraction temperature 46.8 °C, ultrasound time 42.3 min, pH 4.28, and ultrasound power 311 W. Under these conditions, the experimental yield of CEP was 5.98%, which was well matched with the value predicted by the model. Three major polysaccharide fractions (EP-1, EP-2 and EP-3) were obtained by ultrafiltration, chromatography on DEAE-Sepharose Fast Flow and Sephadex G-100 column. Their chemical–physical characteristics were investigated using chemical methods, gas chromatography (GC) and high-performance size-exclusion chromatography (HPSEC). Biochemical characterization of the polysaccharide fractions was evaluated on the basis of free radical scavenging, ferric-reducing antioxidant power (FRAP), lymphocyte proliferation, macrophage phagocytosis and NK cell activity assay in vitro. The results showed that all fractions contained higher galacturonic acid (GalA) and galactose (Gal), possessed considerable antioxidant activity, and could promote proliferation of splenic lymphocytes, activate macrophages and enhance NK cells’ activity. UCE as a more effective and environment-friendly technique could be employed for extracting active ingredients from plant materials, extensively applied in pharmaceutical and food industries.     

On-column refolding of denatured lysozyme by the conjoint chromatography composed of SEC and immobilized recombinant DsbA

DsbA (disulfide bond formation protein A) located in the periplasm of Escherichia coli is a disulfide isomerase, which is vital to disulfide bonds formation directly affecting the nascent peptides folding to the correct conformation. In this paper, recombinant DsbA was firstly immobilized onto NHS-activated Sepharose Fast Flow gel. Then Sephadex G-100 gel was sequentially packed on the top of recDsbA Sepharose Fast Flow, and a so-called conjoint chromatography column composed of SEC and immobilized recombinant DsbA was constructed. Denatured lysozyme was applied on the conjoint column. The effect of SEC volume, flow rate, loading amount and volume, pre-equilibrium mode and KCl concentration in the buffer on lysozyme refolding were investigated in detail and the stability of DsbA immobilization was evaluated. Finally the reusability of the conjoint refolding column was also tested. When loading 2.4 mg denatured lysozyme in 0.5 ml solution, the activity recovery reached 92.7% at optimized experimental conditions, and the conjoint column renaturation capacity decreased only 7.7% after six run reuse due to the use of SEC section in the chromatographic refolding process. The conjoint chromatography offers an efficient strategy to refold proteins in vitro with high productivity and column reusability.