ISOLATION,PURIFICATION, AND N-TERMINAL PARTIAL SEQUENCE OF AN ANTITUMOR PEPTIDE FROM THE VENOM OF THE CHINESE SCORPION BUTHUS MARTENSII KARSCH

ABSTRACT

An antitumor peptide (ANTP) was isolated and purified from the venom of the Chinese scorpion Buthus martensii Karsch. The purification procedure included gel filtration on Sephadex G-50 and Superdex 30 high resolution chromatography, Phenyl Sepharose 6 Fast Flow chromatography, and SP-Sepharose Fast Flow chromatography. Its homogeneity was demonstrated by size exclusion HPLC on TSK G2000 SW. The isoelectric point is more than 10 by pH 3–10 range isoelectric focusing. ANTP has a relative molecular mass of 6280, calculated from the measurement of 16.5% SDS-PAGE. The pharmacological tests showed that ANTP has antitumoral effects in the mouse S-180 fibro sarcoma model and Ehrlich ascites tumor model. Amino acid analysis suggested the ANTP is rich in glycine and does not have histidine and threonine. The sequence of the first 25 N-terminal residues is as follows: Val-Arg-Asp-Gly-Tyr-Ile-Ala-Asp-Asp-Lys-Asn-Cys-Ala-Tyr-Phe-Cys-Gly-Arg-Asn-Ala-Tyr-Cys-Asp-Asp-Glu.     

枯草芽孢杆菌中性β-甘露聚糖酶的纯化及性质研究

经硫酸铵分级沉淀、超滤浓缩、阴离子交换层析和凝胶过滤层析,由枯草芽孢杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）ＢＭ９６０２培养滤液得到了常规凝胶电泳一条带的中性β－甘露聚糖酶．该酶具有与其它已知同类酶相类似的性质,但用ＳＤＳ－ＰＡＧＥ测得该酶分子量为３７ｋＤ;用聚丙烯酰胺等电聚焦电泳测得等电点ｐＩ为４．９．     

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.     

Isolation, purification and characteristics of R-phycoerythrin from a marine macroalga Heterosiphonia japonica

R-phycoerythrin is one of the three phycobiliproteins which are extensively employed as fluorescent probes, and it is prepared from red macroalgae. Phycobiliproteins in the marine red macroalga Heterosiphonia japonica were extracted in 50 mM phosphate buffer (pH 7.0) and precipitated by salting-out. The R-phycoerythrin was isolated by gel filtration with Sepharose CL-4B and Sephadex G-200. Then it was purified by ion exchange chromatography on DEAE Sepharose Fast Flow which was developed by linear ionic strength gradients. The purified R-phycoerythrin gave a ratio of A₅₆₅ to A₂₈₀ of 4.89. It showed a single band and a pI of 4.8 on the examination by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. The polypeptide analysis of the purified R-phycoerythrin by SDS–PAGE demonstrated that it contains four chromophore-carrying subunits and no colorless polypeptide and has two hexameric aggregates. The preparative procedures of the R-phycoerythrin purification established based on the experiments exhibit advantages and can offer a reference for R-phycoerythrin preparation from other marine red macroalga.     

Cyclomaltodextrin glucanotransferase from Bacillus circulans E 192. I. Purification and characterization of the enzyme.

The cyclomaltrodextrin glucanotransferase (CGTase) [1,4-alpha-D-glucan:4-alpha-D-(1,4-alpha-D-glucano)-transferase (cyclizing), EC 2.4.1.19] from Bacillus circulans E 192 has been purified to homogeneity by Cetavlon treatment, ammonium sulfate precipitation, DEAE Trisacryl M chromatography, Q Fast Flow chromatography, and affinity on beta-cyclodextrin-Sepharose 4B. Two isoenzymes were separated by FPLC on a Mono Q column. Their isoelectric points were estimated as 6.7 and 6.9 and they represented 13 and 87%, respectively, of the initial activity. Their molecular weight, pH, and temperature optima were estimated as 78,000, 5.5, and 60 degrees C, respectively. Kinetic parameters indicated that both enzymes had the same properties; they preferentially modified high-molecular-weight substrates to produce cyclodextrins. The apparent Vmax and Km values for soluble starch were 43 mumol of beta-cyclodextrin/min/mg of protein and 0.57% (w/v), respectively. Although this CGTase is not markedly thermostable, it is protected against heat denaturation by substrate, product, and/or calcium ions. The ratios of alpha-, beta-, and gamma-cyclodextrins produced have been determined as 1/7/2 in the initial phase of the reaction and 3/3/1 at equilibrium.     

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.     

Purification and physicochemical properties of different polysaccharide fractions from the water extract of Boschniakia rossica and their effect on macrophages activation

Today more and more attentions had been attracted by many nutritionists and pharmacologists on polysaccharides from natural plants or animals due to their significant biological activities. In this research three polysaccharides (BRR-W1, BRR-WA1 and BRR-WA2) were isolated and purified from the water extract of Boschniakia rossica by DEAE Sepharose Fast Flow and Sepharose 6 Fast Flow column chromatography. Chemical and physical characteristics of three polysaccharides were investigated by a combination of chemical and instrumental analysis methods. The assays of their effect on macrophages activation were also investigated in vitro, including phagocytosis of macrophages, detections for NO production and TNF-α secretion. The results indicated that the effect of polysaccharides on macrophages activation was influenced by their respective physicochemical properties.     

少根根霉(Rhizopus arrhizus)脂肪酶基因在毕赤酵母中的分泌表达

利用PCR技术从少根根霉中扩增出脂肪酶基因(包括前导序列和成熟肽),并将其连接到酵母分泌表达载体pPIC9K中,转化毕赤酵母GS115。利用抗生素G418从重组阳性克隆中筛选得到高拷贝的转化子。在5 L的发酵罐中,当碳源耗尽后开始流加甲醇诱导脂肪酶的表达,经过96 h培养后发酵液上清液中重组脂肪酶(rRAL)的表达量约为90 mg/L。rRAL经过超滤,SP-Sepharose离子交换层析和Butyl-Sepharose疏水层析纯化。纯化后的蛋白在SDS-PAGE上为单一条带,表观分子量为32 kDa,比酶活为1 543 U/mg。N-端序列分析表明rRAL是经过加工后的产物。同时没有发现全长的Rhizopus arrhizus脂肪酶(RAL)被分泌表达。     

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。     

Purification and properties of a beta-galactoside-binding lectin from neonatal marmoset.

A beta-galactoside-binding lectin was extracted from whole neonatal marmoset homogenate with lactose solution and purified to homogeneity by ion-exchange chromatography on Q Sepharose Fast Flow and by affinity adsorption to trypsinized and glutaraldehyde-fixed ghosts of rabbit erythrocytes. The lectin has a dimeric structure composed of two 15K subunits. Its amino acid composition and partial amino acid sequences were quite similar to those of beta-galactoside-binding lectins from human placenta and lung.     

Generic/matrix evaluation of SV40 clearance by anion exchange chromatography in flow-through mode

The potential of viral contamination is a regulatory concern for continuous cell line-derived pharmaceutical proteins. Complementary and redundant safety steps, including an evaluation of the viral clearance capacity of unit operations in the purification process, are performed prior to registration and marketing of biotechnology pharmaceuticals. Because process refinement is frequently beneficial, CBER/FDA has published guidance facilitating process improvement by delineating specific instances where the bracketing and generic approaches are appropriate for virus removal validation. In this study, a generic/matrix study was performed using Q-Sepharose Fast Flow (QSFF) chromatography to determine if bracketing and generic validation can be applied to anion exchange chromatography. Key operational parameters were varied to upper and lower extreme values and the impact on viral clearance was assessed using simian virus 40 (SV40) as the model virus. Operational ranges for key chromatography parameters were identified where an SV40 log(10) reduction value (LRV) of >or=4.7 log(10) is consistently achieved. On the basis of the apparent robustness of SV40 removal by Q-anion exchange chromatography, we propose that the concept of "bracketed generic" validation can be applied to this and potentially other chromatography unit operations.     

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.     

S-腺苷甲硫氨酸合成酶的组成型表达、产物纯化及鉴定

将大肠杆菌(E.coli K12) S 腺苷甲硫氨酸合成酶(SAMS)基因克隆至质粒pBR322中,获得的重组质粒pBR322-SAMS转入大肠杆菌JM109菌株,构建了能高效组成型表达SAMS的重组菌E.coli JM109 (pBR322-SAMS)。将重组大肠杆菌破碎后上清液经20%~40%硫酸铵分级盐析、Phenyl-Sepharose Fast Flow疏水层析和Q Sepharose Fast Flow离子交换层析,即可得到纯度提高5倍,比活为48.7 μ/mg的SAMS,三步纯化的总回收率为62%,纯度达到92%。SAMS表达量为1 176μ/L,占到菌体可溶性总蛋白的20%。重组酶的最适反应pH为8.5,4℃下在pH 7.5的缓冲液中保温10h酶活性几乎不改变。重组酶反应的最适温度为55℃ ,酶活性稳定的温度范围为20~35℃。重组酶的KmL Met为0.22mmol/L,Vmax L-Met为1.07mmol/(L·h),Km ATP为0.52 mmol/L,Vmax ATP为1.05 mmol/( L·h)。     

四季豆种子中两种蛋白质的分离纯化及其性质初步研究

四季豆（ＰｈａｓｅｏｌｕｓｖｕｌｇａｒｉｓＬ．）属于豆科蝶形花亚科野百合属,在我国分布极广,普植于温带地区．虽然四季豆是一种极普通的营养作物,但贮藏过久或煮沸不透常发生中毒．四季豆种子中成分复杂,除了蛋白组分外,还含有多种小分子组分［１］．一般认为,四季豆种子的毒性是这些组分复合作用的结果一直引人关注的则是其中的有毒蛋白．四季豆蛋白中很多是凝集素［２～４］,四季豆凝集素是一种典型的植物血细胞凝集素（Ｐｈｙｔｏｈｅｍａｇｇｌｕｔｉｎｉｎ,ＰＨＡ）,是一种寡糖结合专一性的糖蛋白,具有凝聚血细胞,刺激…     

Purification Process for the Preparation and Characterizations of Hen Egg White Ovalbumin,Lysozyme, Ovotransferrin,and Ovomucoid

Abstract

In this study, four major egg white proteins were purified by two step ion exchange chromatography followed by precipitation. Lysozyme and ovalbumin were separated with Q Sepharose Fast Flow anion exchange chromatography in the first step, resulting in two peaks of lysozyme and three peaks of ovalbumin with 87% and 70% purity by HPLC, respectively. Ovotransferrin was separated with CM-Toyopearl 650 M cation exchange chromatography in the second step, giving 80% purity. Ovomucoid was precipitated from the partial purified protein fraction from the first two steps, and concentrated in the final step to yield 90% purity. Protein recoveries were estimated to be 55, 21, 54, and 21% for lysozyme, ovotransferrin, ovalbumin, and ovomuciod, respectively. Lysozyme was identified from the purified peaks using zymogram refolding gel, whereas ovalbumin was identified by western blotting. Purified ovotransferrin and ovomucoid was identified by mass spectrometry. The results indicate that this purification process is adequate for preparation of lysozyme, ovalbumin, ovotransferrin, and ovomucoid, at least on a laboratory scale.     

Zinc-binding proteins from boar seminal plasma -- isolation, biochemical characteristics and influence on spermatozoa stored at 4°C

Affinity chromatography on Chelating Sepharose Fast Flow Gel-Zn(2+) was used for fractionation of boar seminal plasma proteins. Approximately 30% of total boar seminal plasma proteins showed affinity for zinc ions (ZnBP fraction). Native electrophoresis (PAGE) of ZnBP revealed six protein fractions which separated into 27 bands under denaturing conditions (SDS/PAGE). Two-dimensional electrophoresis (2D PAGE) showed 148 polypeptides with isoelectric points mostly in the basic and neutral pH range. The zinc-binding proteins comprise mainly 10-20 kDa polypeptides which are probably members of the spermadhesin family. ZnBP present in the incubation mixture of spermatozoa stored for 1 or 24 h at 4 °C allowed preservation of a higher percentage of cells exhibiting linear motility in comparison to a control sample stored in PBS. Presented results indicate that proteins binding Zn(2+) ions have a shielding effect on the sperm plasma membrane and acrosome of spermatozoa, protecting these structures against consequences of cold shock.     

重组腺病毒Ad-GFP的纯化

目的：用色谱法纯化制备携带绿色荧光蛋白基因的重组腺病毒。方法：采用5L生物反应器悬浮培养HEK-293N3S细胞进行病毒的扩增,冻融法裂解细胞,通过超滤、Source15Q离子交换层析和Sepharose4Fast Flow凝胶过滤层析进行分离纯化,采用分光光度法和高效液相色谱法分析重组腺病毒产品的纯度,采用组织培养半数感染剂量方法测定病毒的感染滴度。结果：通过两步色谱层析得到产品,经分光光度法分析,其D260nm/D280nm比值为1.21,高效液相色谱法分析其纯度达96.5%,产品滴度为1.0×1011U/mL,病毒颗粒数为1.76×1012/mL,比活性为5.68%。结论：确定了稳定可行的重组腺病毒色谱分离工艺,得到的产品在纯度、滴度和比活性等方面均符合国家食品药品监督管理局的标准。     

Purification of TAXI-like Endoxylanase Inhibitors from Wheat (Triticum Aestivum L.) Whole Meal Reveals a Family of Iso-forms

An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N -hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase.     

Purification of TAXI-like endoxylanase inhibitors from wheat (Triticum aestivum L.) whole meal reveals a family of iso-forms

An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N-hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase.     

Purification of papain by immobilized metal affinity chromatography (IMAC) on chelating carboxymethyl cellulose

Chelating carboxymethyl cellulose was prepared in bead form by immobilizing iminodiacetic acid on carboxymethyl cellulose which was earlier crosslinked and activated by epichlorohydrin. The prepared matrix was used to purify papain by a factor of 2.6 from commercial papain, and by a factor of 4 from papaya latex by batch adsorption and immobilized metal affinity chromatography respectively. Purification factors obtained were equal in batch mode and double in column mode, to purifications obtained on Chelating Sepharose® Fast Flow. Flow rates up to 38 ml/cm2 h were easily possible on the prepared chelating carboxymethyl cellulose.