两步柱色谱法分离纯化重组肿瘤血管生长抑制因子Kringle 5

建立了一种用Ni2+螯合的Chelating Sepharose Fast Flow亲和柱色谱和Sephadex G-75凝胶排阻柱色谱分离纯化重组肿瘤血管生长抑制因子Kringle 5的方法。采用该工艺得到的重组Kringle 5经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析表明其纯度约为98%,且具有抑制鸡胚绒毛尿囊膜新生血管生成的生物活性。     

基因重组可溶性非融合血管生成抑制剂Kringle 5的色谱分离和纯化

Kringle 5是血纤维蛋白溶酶原中特异抑制内皮细胞增生和迁移活性最高的一种血管生成抑制剂。该实验在前期成功克隆和表达可溶性非融合血管生成抑制剂Kringle 5的基础上,建立了一种两步色谱法分离纯化Kringle 5的方法。首先用SP Sepharose Fast Flow强阳离子交换色谱柱对Kringle 5重组菌体破碎上清液进行初步分离,然后再用丙烯葡聚糖凝胶S-100 HR凝胶排阻色谱柱对其进行进一步的纯化。采用本方法得到的可溶性非融合血管生成抑制剂Kringle 5经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和高效凝胶排阻色谱检测其纯度大于98%,通过鸡胚尿囊膜法确定这种蛋白质具有抑制内皮毛细血管生长的活性。     

两步柱色谱法分离纯化重组猪β2-肾上腺素能受体

β2-肾上腺素能受体是细胞表面受体的一种,它能通过偶联异源三聚体G蛋白将信号转导引入到细胞内部。本实验在成功克隆、表达β2-肾上腺素能受体的基础上,建立了一种两步柱色谱分离纯化目的蛋白质的方法。首先利用Ni2+螯合的高分辨纯化的预带电荷介质Sepharose High Performance与含有六聚组氨酸标签的蛋白质特异结合的性质,对目的蛋白质进行初步分离,接着运用快流速Q琼脂糖凝胶(Quaternary Sepharose Fast Flow)对其进行进一步的分离纯化。采用该方法得到的β2-肾上腺素能受体蛋白质经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和高效凝胶排阻色谱检测其纯度约为95%。结果表明该方法可以对重组猪β2-肾上腺素能受体进行有效的分离纯化。     

增强型绿色荧光蛋白的色谱分离和纯化

增强型绿色荧光蛋白(EGFP)是生物领域常用的标记物。在前期成功克隆表达EGFP的基础上,本实验建立了两步分离纯化EGFP的色谱方法,并验证其分离纯化效果,检验EGFP的活性。首先用金属螯合亲和色谱柱HisTrap HP对EGFP的重组菌体破碎上清液进行初步分离,再用葡聚糖凝胶排阻色谱柱Sephadex G-10 HR对其进行脱盐纯化。采用丙烯葡聚糖凝胶排阻色谱柱Sephacryl S-300 HR和十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测分离纯化后的EGFP纯度。最后通过荧光分光检测器和非变性聚丙烯酰胺凝胶电泳(Native-PAGE)验证分离纯化后的EGFP是否具有荧光活性。结果表明该方法可以简便快速地分离纯化EGFP,纯度超过98%,同时保持了EGFP的荧光活性。     

一种从蛇毒中纯化神经生长因子的新工艺

为了能从中华眼镜蛇蛇毒中简单快速地分离纯化神经生长因子（一种治疗各种神经性损伤和神经退行疾病的药物,简称NGF）,采用不同的色谱柱联用的方式对NGF的纯化工艺进行了研究。结果表明,采用DEAE Sepharose F.F.和Sephadex G 50二步柱色谱工艺可以从蛇毒中快速分离得到神经生长因子。经十二烷基硫酸钠聚丙烯酰胺凝胶电泳和反相高效液相色谱分析, 证明所得到的NGF为单一组分,相对分子质量约为 29 000。对8　d龄鸡胚背根神经节采用体外培养法,结果证明,所得NGF具有明显的促进神经纤维     

钳蝎毒中抗癫痫肽、镇痛肽和抗肿瘤肽的快速同时分离和鉴定

采用Shim-Pack WCX-1型阳离子交换高压色谱柱对中国东亚钳蝎全蝎毒进行了分离，在鉴定了其中的抗癫痫肽、镇痛肽和抗肿瘤肽活性峰的基础上，应用Shim-Pack DIOL-300型凝胶排阻高压色谱柱对它们进行了进一步分离和鉴定，可以得到较纯的3种多肽。在高压色谱所提供的全蝎毒分离信息的基础上，应用与Shim-Pack WCX-1色谱柱具有相同交换基团的、具有较大吸附容量的CM Sepharose CL-6B软胶介质在低压色谱上对全蝎毒进行了分离，并分别对其中的抗癫痫肽、镇痛肽和抗肿瘤肽进行了鉴定。     

基于特征肽的超高效液相色谱-串联质谱法检测矛头蝮蛇蛇毒种属来源及类凝血酶含量

蛇毒血凝酶类药物是以蝮蛇蛇毒为原料制备的止血药,主要活性成分为蛇毒类凝血酶(svTLEs)。不同蛇种来源的svTLEs结构不同,止血机制不同,药理作用也存在差异,因此准确鉴别蛇毒种属来源和svTLEs含量对于保障该类产品的质量至关重要。研究基于蛋白质组学技术,筛选出了具有种属特异性的矛头蝮蛇svTLE特征肽,并建立了基于特征肽的超高效液相色谱-串联质谱(UHPLC-MS/MS)检测矛头蝮蛇蛇毒种属来源及类凝血酶含量的方法。采用胰蛋白酶对纯化的矛头蝮蛇svTLE进行酶解,利用纳升液相色谱-四极杆/静电场轨道阱高分辨质谱(Nano LC-Q-Exactive-MS)和Proteome Discoverer 2.2软件分别进行多肽的检测和鉴定,通过BLAST搜索与Uniprot数据库对比分析,筛选出具有种属特异性的矛头蝮蛇svTLEs特征肽“EAYNGLPAK”。针对该特征肽对酶解温度、酶解时间和酶用量等样品前处理方法进行了优化,利用超高效液相色谱-串联质谱,以m/z 481.9>315.2和481.9>485.2作为检测离子对,采用ESI⁺模式进行了多反应...     

强阴离子交换色谱结合分子排阻色谱纯化血清中免疫球蛋白和白蛋白

动物血清中免疫球蛋白和白蛋白的等电点分别约为7.8和4.8,根据它们等电点的较大差别,利用Q SepharoseTM-XL强阴离子交换色谱结合分子排阻色谱同时分离纯化这2种蛋白。以0.02 mol/L pH 8.0的Tris-HCl缓冲液平衡离子交换色谱柱并将已稀释10倍的高免疫的兔血清上样,采用pH分段洗脱。在pH 6.0时以0.3 mL/min低流速洗脱得到高纯度的免疫球蛋白,继续在pH 4.0时洗脱,再辅以Sephadex G-75分子排阻色谱可获得纯度大于95%的白蛋白。对纯化后的蛋白进行活性检测,证明所纯化的免疫球蛋白和白蛋白都保持正常的生物活性。蛋白质含量测定说明免疫球蛋白的纯化回收率达到95%以上,而白蛋白的纯化回收率大于90%。该法简便快速,可同时从动物血清中纯化出保持生物活性的免疫球蛋白和白蛋白,纯化效率高。     

从猪血中分离纯化高纯度的猪血红蛋白

为了从猪血中分离纯化高纯度的猪血红蛋白,建立了通过超滤、DEAE-Sepharose Fast Flow离子交换色谱和Sephadex G-75凝胶 排阻色谱三步法制备高纯度猪血红蛋白的方法,并通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、高效凝胶排阻色谱和反相高 效液相色谱方法,对纯化后的猪血红蛋白进行了鉴定。经三步分离纯化后,猪血红蛋白的纯度大于99%,含量为1.328 g/L。     

高效阳离子交换法分离纯化蛋清中的溶菌酶

 建立了用高效阳离子交换分离纯化蛋清中溶菌酶的新方法 ,讨论了纯化的工艺条件。蛋清样品匀浆后 ,用氯化钠初步纯化 ,然后用弱阳离子交换柱XIDACE WCX分离。结果表明 ,被纯化的溶菌酶和杂蛋白得到很好分离。经活性检测 ,溶菌酶过柱后的活性回收率为 10 7% ,蛋白的比活为 15 4 6 7U/mg ,纯化倍数提高了 5 6倍。用尺寸排阻 (SEC)鉴定 ,得到的溶菌酶呈均一性。该法较传统软基质低压离子交换分离速度快 ,纯化效率高。     

An investigation of the coagulological potential of the venoms of some Central Asian snakes

The compositions of the proteolytic complexes of the venoms of the crotalid Agkistrodon halys halysand the viperid Echis multisquamatushave been investigated with the aid of a large number of specific natural and model substrates. A comparative analysis of the results obtained has revealed a predominance in the crotalid venom of fibrinogen-hydrolyzing enzymes, while the viperid venom is characterized as a procoagulant of the prothrombin-activating type. Among the fibrinogenases of the crotalid venom, a thrombin-like enzyme and a plasmin-like proteinase have been revealed and obtained in a purified state. A prothrombin-activating enzyme from the viperid venom has been isolated in the purified state and characterized with the aid of specific substrates.     

pBPB共价修饰蝮蛇酸性磷脂酶A2的晶体学数据

The Crystals of acidic phospholipase A2 from the venom of Agkistrodon blomhoffii brevicaudus (i.e. Agkistrodon halys pallas) covalently modified with p-bromo-phenacylbromide were obtained by the method of hanging drop vapor diffusion. Crystallization droplet was composed of 0.06mol?L-1 Na(CH3)2AsO2 (pH=6.5), 24.8% (v/v) 1,4-butanediol, and 4.5mg?mL-1 protein. The crystal data are of a=b=82.82?., c=32.85?, space group P61. 8945 unique reflections with 1.93 ? resolution were measured on a Siemens area detector X-ray diffractometor, of which 8069 reflections having F0 >2σ(F). The experimental results show that the crystals are suitable to a structure analysis of high resolution.     

正交晶型江浙蝮蛇毒碱性磷脂酶A2初步结晶学研究

The basic phospholipase A2 from the venom of Agkistrodon halys pallas possesses the ability to cause hemolysis in contrast to the other two phospholipase A2 from the same venom. A new form of crystals of this enzyme was grown. The crystals belong to space group of P212121 with unit cell parameters of a=9.175nm, b=10.080nm , c=2.287nm.The crystals diffract to high resolution, and are suitable for detailed structural studies. The data were collected up to 0.25nm resolution using synchrotron radiation-imaging plate-Weissenberg camera system. Preliminary analysis reveals the presence of two molecules in the asymmetric uint and the molecule may pack with the smallest dimension approximately parallel to the c axis. The new crystal form is more attractive than the monoclinic one previously reported for crystallographic structure determination as it contains fewer molecules in the asymmetric unit.     

An investigation of the coagulological potential of the venoms of some Central Asian snakes

The compositions of the proteolytic complexes of the venoms of the crotalid Agkistrodon halys halysand the viperid Echis multisquamatushave been investigated with the aid of a large number of specific natural and model substrates. A comparative analysis of the results obtained has revealed a predominance in the crotalid venom of fibrinogen-hydrolyzing enzymes, while the viperid venom is characterized as a procoagulant of the prothrombin-activating type. Among the fibrinogenases of the crotalid venom, a thrombin-like enzyme and a plasmin-like proteinase have been revealed and obtained in a purified state. A prothrombin-activating enzyme from the viperid venom has been isolated in the purified state and characterized with the aid of specific substrates. Work performed with the support of the Nederlandse Organisatie voor Wetenschappelijk Onderzoek.Institute of Biochemistry of the Academy of Sciences of the Uzebekistan Republic, Tashkent. Department of Biochemistry, Cardiovascular Research Institute, University of Limburg, Maastricht, The Netherlands. Translated from Khimiya Prirodnykh Soedinenii, Vol. 3, pp. 447–454, May–June, 1993.     

分离尿激酶的亲和色谱填料的制备

 合成了分别以 Sepharose和聚甲基丙烯酸环氧丙酯为基质、对氨基苯甲脒为配基的分离尿激酶的两种亲和色谱填料 ,并用于尿激酶粗品的直接纯化 ,活性回收率分别为 1 0 8.3 %和 43 .4% ,比活提高倍数分别为 9.0 6倍和 3 6.9倍。     

High-capacity purification of hen egg-white proteins by ion-exchange electrochromatography with an oscillatory transverse electric field

The ion-exchange electrochromatography with an oscillatory electric field perpendicular to the mobile-phase flow driven by pressure (pIEEC) was used to separate hen egg-white (HEW) proteins. The results were compared with those of normal ion-exchange chromatography (IEC). The column was designed as three-compartment rectangular column of 2-mL with dimensions (length x width x depth) of 40 x 10 x 5 mm(3) and the electric field was applied across the direction of column width. Q Sepharose FF was packed into the central compartment as the chromatographic bed. It was confirmed that the dynamic binding capacity (DBC) of different proteins (ovotransferrin and ovalbumin) in the HEW solution increased 2.3 times when an oscillatory electric current of 30 mA at 1/20 Hz was applied in the transverse column direction. Then, the HEW proteins were separated by the pIEEC at loading amounts 2.3-fold higher than those by the IEC. When the feedstock of about one-third of the DBC was applied to the columns (i.e., 7 mL for the pIEEC and 3 mL for the IEC), similar separation efficiencies of the two chromatographic modes were achieved. Both the recovery yield and purity reached 73% to over 90%. The results indicate that the pIEEC is promising for high-capacity purification of proteins.     

阴离子交换整体柱对蛋白质的分离与纯化

分别用乙二胺、二乙胺、三乙胺将自制的以甲基丙烯酸缩水甘油酯(GMA)为单体、乙二醇二甲基丙烯酸酯(EDMA)为交联剂的整体柱修饰为弱、强阴离子交换整体柱。考察了该整体柱的性能,选择出分离蛋白质(牛血清白蛋白、溶菌酶和谷胱甘肽)的最佳实验条件,并在最佳分离条件下考察了这些蛋白质在整体柱上的色谱行为和该整体柱对纤维素降解酶的分离纯化情况。实验结果表明,该整体柱性能良好,可以实现对纤维素降解酶的快速分离与纯化。同时,实验也证明采用梯度洗脱可以实现对某些蛋白质的分离纯化。     

Rapid displacement chromatography of melittin on micropellicular octadecyl-silica.

Rapid high-performance liquid chromatographic analysis and displacement purification of melittin and its variants were carried out by reversed-phase chromatography. High speed of separation was achieved by the use of columns packed with a micropellicular stationary phase consisting of a thin C18 hydrocarbonaceous layer on the surface of 2-microns fluid-impervious silica microspheres at elevated temperature. In the case of melittin from bee venom or its synthetic variants the plots of the logarithmic retention factor against acetonitrile concentration in the eluent were straight lines whereas the van't Hoff plots in the temperature range from 20 to 80 degrees C were non-linear. Purification of melittins by displacement was carried out with benzyldimethylhexadecyl ammonium chloride as the displacer. In a 20-min displacement run at 40 degrees C about 5 mg of highly pure melittin were isolated from 10 mg of synthetic mixture by using a 105 x 4.6 mm column. The results demonstrate that columns packed with micropellicular sorbents not only facilitate rapid high-performance liquid chromatographic analysis but are also suitable for fast peptide purification with high recovery.     

Modeling the effects of column packing quality and residence time changes on protein monomer/aggregate separation

The packing quality of chromatography columns used for the purification of protein therapeutics is routinely monitored to ensure consistent and reproducible performance. In this work, we used established chromatography models to determine the effect of column packing quality and fluid residence time on the separation of protein therapeutic monomer and aggregate species using a hydrophobic interaction chromatography adsorbent (Phenyl Sepharose Fast Flow). The relationship between the number of theoretical plates, fluid residence time, and column separation performance was quantified using modeling simulations. The simulations showed the separation depended on both the fluid residence time and the number of theoretical plates. However, when the number of theoretical plates was increased to ≥150, the simulations predicted that the separation performance of the column was not significantly improved. The approach described here could be used as a method to quantify acceptable height equivalent of a theoretical plate values for columns, and serve as a tool to understand how column packing quality impacts a given chromatographic separation prior to column scale-up, as well as during the monitoring of column lifetime in the manufacturing of large scale protein therapeutics.