依赖雄激素的大鼠储精囊分泌蛋白的离子交换层析纯化

本文报道利用阳离子交换层析,纯化了雄激素依赖的大鼠储精囊分泌蛋白(SVPⅡ、SVPⅣ、SVPⅤ_a、SVPⅤ_b及SVPⅥ)。主要纯化步骤包括下列二步:1.Sep-hadex G-100凝胶过滤;2.上样后,联合使用盐梯度和pH梯度,洗脱快速蛋白液相层析(FPLC)系统的阳离子交换柱Mono S。洗脱峰的纯度以变性条件下的聚丙烯酰胶凝胶电泳(SDS-PAGE)和等电聚焦(IEF)鉴定;借此,还测定了已纯化的大鼠储精囊分泌蛋白的分子量和等电点。     

白菜型油菜种子胰蛋白酶抑制剂纯化及部分性质研究

采用热变性、硫酸铵分步盐析及离子交换层析和分子筛层析等方法,从白菜型油菜种子中得到胰蛋白酶抑制剂(BNTI)。SDS-PAGE检测为单一条带,表明纯化的胰蛋白酶抑制剂电泳均一。SDS-PAGE测定其分子量约为14．4kD,等电聚焦测定其等电点约为4．7。BNTI具有较高的热稳定性。本文还考察了温度对溶液中BCH蛋白构象的影响,荧光光谱和测定抑制活力结果表明BNTI中的色氨酸和酪氨酸残基位于疏水部位。     

铁蛋白分析技术的研究进展

铁蛋白是含铁蛋白质,1943年Cranick用硫酸铵沉淀、超滤、硫酸镉重结晶、凝胶层析等技术,获得铁蛋白,经免疫电泳、等电聚焦电泳和离子交换层析鉴定,血清铁蛋白由含铁单体,     

层析法纯化破伤风毒素的试验研究

为了获得高纯度的破伤风毒素,用疏水层析和离子交换层析纯化破伤风毒素。破伤风毒素培养滤液经Phenyl Sepharose疏水层析除去大部分杂质,再经DEAE Sephadex离子交换层析进一步纯化。经两步层析纯化后,毒素纯度达到2000Lf/mg PN以上,回收率为52%~73%。用此方法,连续纯化五批毒素,均获得高纯度的破伤风毒素。试验证明破伤风毒素经疏水层析和离子交换层析可得到有效纯化。     

人前列腺生长因子的分离纯化及生物活性鉴定

从人良性增生前列腺组织中经硫酸铵沉淀和肝素－琼脂糖凝胶层析纯化出人前列腺生长因子（ｈＰＧＦ）,纯化倍数约１０００倍,ＳＤＳ－ＰＡＧＥ和等电聚焦电泳示分子量约为１７ｋＤ、等电点同标准ｂＦＧＦ,利用分离培养的人前列腺间质成纤维细胞进行活性鉴定,发现以１．３～１．７ｍｏｌ／ＬＮａＣｌ洗脱部分为ｈＰＧＦ,活性最高,对间质成纤维细胞有显著刺激增殖作用。     

人尿激肽原酶的纯化与鉴定

采用两性离子胶体沉淀和乙醇沉淀相结合的粗提方法,经离子交换、疏水层析、亲和层析及凝胶过滤4个步骤有效地将人尿激肽原酶（hk-1）粗提物纯化,比活提高了1 755倍,总得率为70%.用以慈菇蛋白酶抑制剂为配体的亲和层析纯化hk-1,效果理想,整个工艺路线适合产业化生产.纯化产物在SDS-聚丙烯酰胺凝胶电泳上为单带,高压液相色谱(HPLC)上为单峰,基质辅助激光解析电离飞行时间质谱测得分子质量为33 450 u,等电聚焦测得pI在4.3附近,为含糖蛋白.同时测定了该酶的热稳定性和pH稳定性.纯化过程中同时分离得到另一种药用蛋白——人尿胰蛋白酶抑制剂（HUTI）.     

葡萄球菌肠毒素的研究进展

葡萄球菌肠毒素是引起细菌性食物中毒的主要原因之一,葡萄球菌肠毒素按照血清学的方法可分为SEA、SEB、SEC1-3、SED和SEE等七个经典肠毒素。但仍有5％未知的新型肠毒素存在,其编码基因有seg、sei、sej、sek、sel等。葡萄球菌肠毒素也是一种超抗原,能刺激非特异性T细胞的大量增殖。本文对葡萄球菌肠毒素结构、编码基因和功能等方面的研究进展作了简要综述。     

人前列腺生长因子的分离纯化及生物活性鉴定

从人良性增生前列腺组织中经硫酸铵沉淀和肝素－琼脂糖凝胶层析纯化出人前列腺生长因子（ｈＰＧＦ）,纯化倍数约１０００倍,ＳＤＳ－ＰＡＧＥ和等电聚焦电泳示分子量约为１７ｋＤ、等电点同标准ｈＦＧＦ。利用分离培养的人前列腺间质或纤维细胞进行活性鉴定,发现以１．３￣１．７ｍｏｌ／Ｌ ＮａＣｌ洗脱部分为ｈＰＧＦ,活性最高,对间质成纤维细胞有显著刺激增殖作用。     

重组双功能水蛭素的发酵、纯化和鉴定

构建了重组双功能水蛭素 ( Recombinant-RGD-Hirudin、 r-RGD-Hirudin ) cDNA 的表达质粒 RGD-Hirudin-pPIC9K,转化入毕赤酵母中,经筛选得到高表达的阳性克隆。种子菌经过3d发酵培养,其培养液上清经超滤浓缩、凝胶过滤层析和离子交换层析后,得到纯度大于97%、比活性为 12000 ATU/mg 的 r-RGD-Hirudin,回收率大于60%,发酵产率为 1 g/L。纯化后的 r-RGD-Hirudin 经过还原SDS-PAGE,抗凝血酶活力分析、抗血小板聚集分析、质谱分析及等电聚焦分析等方法鉴定,证明该表达产物为水蛭素的衍生物,具有抗凝血酶和抗血小板聚集双重功能。     

菜心叶片中乙醇酸氧化酶的多种组分

乙醇酸氧化酶（EC．1．1．3．1,GO）是光呼吸中的关键酶,过去对其电泳行为和等电点的报告互不一致。Grodzinski和Col－man（1972）将菠菜、烟草等植物部分纯化的GO酶液在pH8．3的聚丙烯酰胺凝胶中电泳,活性染色后出现两条活性带,两者对FMN的依赖性有一定差异。Kerr和Groves（1975）对豌豆叶片部分纯化的GO酶液进行等电聚焦电泳,表明其pI值大于9．6.Behrends等（1982）将绿色黄瓜子叶的酶液经聚焦层析,发现GO活性分布在pH8．7附近。但Nishimura等（1983）发现在pH8．9的凝胶中南瓜子叶GO不迁移,而在pH4．5的凝胶中则出现一…     

Studies on phosphoribosylpyrophosphate synthetase from Giardia intestinalis.

A substantial degree of purification, up to 3200-fold, with recoveries of 8-11% of phosphoribosylpyrophosphate (P-Rib-PP) synthetase from Giardia intestinalis extracts was achieved by the high resolution techniques of anion exchange chromatography and chromatofocusing columns on a fast protein liquid chromatography system. A Mono P chromatofocusing column gave rise to an enzyme peak eluting from the column at pH 4.5, indicating that the enzyme has an isoelectric point (pI) at approximately this value. The molecular weight of the enzyme was found to be 150,000 from a Sephacryl S-200 column. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified enzyme gave a single protein band with a subunit molecular weight of 38,000, indicating that the enzyme existed as a tetramer. The properties of G. intestinalis P-Rib-PP synthetase in terms of pH optimum, isoelectric point, subunit structure, phosphate requirement, metal and nucleotide specificity, appear to be very similar to those of the enzyme from other sources.     

Purification and partial characterization of the extracellular gamma-D-glutamyl-(L)meso-diaminopimelate endopeptidase I, from Bacillus sphaericus NCTC 9602

The gamma-D-glutamyl-(L)meso-diaminopimelate endopeptidase, or endopeptidase I, from Bacillus sphaericus 9602 was purified to apparent protein homogeneity. The purification was achieved by a six-step procedure: ammonium sulfate fractionation, phenyl-Sepharose chromatography, two consecutive DEAE-Trisacryl chromatographies, chromatofocusing and Sephacryl S-200 permeation chromatography. The enzyme was purified 5000-fold with a 38% recovery of lytic activity. It is an acidic protein (pI 5.4) of hydrophobic nature. Kinetic studies have shown a Km value of 0.57 mM and an apparent Vmax of 8.3 mumol min-1 (mg enzyme)-1 with N-acetylmuramyl-L-alanyl-gamma-D-glutamyl-(L)meso-diaminopimelyl (L)-D-[14C]alanine as substrate. The enzyme was inhibited by o-phenanthroline and EDTA and was reactivated by zinc, cobalt and manganese ions; thus endopeptidase I is a metallo enzyme, probably a zinc enzyme. Moreover it is a heat-stable protein with an apparent inactivation temperature of 80 degrees C.     

Purification and characterization of streptococcal erythrogenic toxin type A produced by Streptococcus pyogenes strain NY-5 cultured in the synthetic medium NCTC-135. Comparison with the dialyzed medium (TP medium)-derived toxin

Streptococcal erythrogenic toxin type A (ET-A) was purified from culture filtrate of Streptococcus pyogenes strain NY-5 grown in a chemically defined synthetic medium NCTC-135. We succeeded in simplifying the purification procedure, and obtained a highly purified preparation of ET-A. The purification procedure was the combination of ultrafiltration with Amicon PM-10 and YM-10 membranes, chromatofocusing with PBE-94 exchanger (pH 4.0-6.0), and gel filtration through Sephacryl S-200. The purified toxin protein showed a single band with Mr 28,000 on SDS-PAGE and had pI 5.2 on agarose IEF. HPLC chromatography pattern of the toxin revealed one symmetric peak. The result of amino acid analysis of the toxin was in accordance with that of Gerlach et al and with Weeks and Ferretti who reported the nucleotide sequence of the spe A gene. Biological activities of the purified toxin were remarkably potent. The mitogenic activity for rabbit lymphocytes and one skin test dose in rabbit were found at the lower dose of 10 pg and 1 ng of the toxin, respectively.     

Purification and characterization of 3-dehydroquinase from Escherichia coli.

A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli. Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.     

Purification and characterization of histidine decarboxylase from mouse kidney.

Histidine decarboxylase was purified 800-fold from the kidneys of thyroxine-treated mice. The purification procedure included precipitation of protein from a crude supernatant after heating it to 55 degrees C at pH 5.5, fractionation with (NH4)2SO4, phosphocellulose column chromatography, chromatofocusing, DEAE-Sepharose column chromatography, gel filtration on Sephacryl S-300 and preparative polyacrylamide-gel electrophoresis. The native enzyme had an estimated Mr of 113 000. The protein was analysed in SDS/10%-polyacrylamide gels and formed a single band corresponding to a subunit Mr of 55 000, indicating that it is a dimer. Three forms of the enzyme were resolved on isoelectrofocusing gels, with pI 5.3, 5.5 and 5.7.     

Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins.

Cathepsin S was detected in bovine kidney, spleen, lymph nodes and lung by immunochemical methods. The immunostaining of cathepsin S in kidney was concentrated to the cells of the proximal tubule, where the enzyme was present in cytoplasmic granules. The purification method for cathepsin S from bovine spleen involved (NH4)2SO4 fractionation, chromatography on CM-Sephadex C-50, gel filtration on Sephacryl S-200 and chromatofocusing (pH 8.0-6.0). The enzyme was partially destroyed by autolysis of the homogenate at pH 4.2. The isoelectric point of cathepsin S was 7.0. Cathepsin S was found to hydrolyse proteins at a similar rate to cathepsin L below pH 7.0. At pH values of 7.0-7.5 cathepsin S retained most of its activity, whereas cathepsin L was completely inactive.     

Purification and properties of the coupling-factor ATPases F1 from Rhodopseudomonas palustris and Rhodopseudomonas sphaeroides

The coupling-factor ATPases from photosynthetically grown Rhodopseudomonas palustris and Rhodopseudomonas sphaeroides were purified by the same procedure to homogeneity. Gel chromatography on Sephacryl S-300 Superfine shortened the process of purification and improved its yield. Solubilization of the ATPase from both bacteria was found to be dependent on a specific sonication treatment of the cell suspensions, indicating a very weakly bound F1-ATPase in R. palustris. Depleted chromatophores could be restored in photophosphorylation and membrane-bound ATPase activities by adding the solubilized ATPase protein. The purified enzymes did not show a markedly trypsin-stimulated or dithiothreitol-stimulated activity. Isoelectric focusing and chromatofocusing revealed isoelectric points of 5.0 for both F1-ATPases. The molecular weights were determined by gel chromatography plus high-performance liquid chromatography. Hence, we calculated a molecular weight of 350000 for both F1-ATPases. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed five subunits for both enzymes. Kinetic parameters, regarding substrate specificity, the effect of divalent cations, Km and Ki values for the membrane-bound and solubilized ATPases were determined.     

重组尿激酶原的纯化和性质研究

CHO工程细胞11G持续表达的pro-UK分泌在细胞培养液的上清中,培养液上清经过微孔玻璃(MPG)吸附色谱,羧甲基阳离子交换色谱,高压液相凝胶色谱三步纯化,纯化倍数可达700倍以上,总回收率为46％．再经过Benzamidine-Sepharose 6B亲和层析去掉少量的双链尿激酶,得到纯化尿激酶原．终产物经SDS-PAGE银染分析,纯度达90％以上,分子量为52 ku,其比活性为51 220 U/mg．抗体中和、二异丙基氟磷酸(DFP)抑制等实验证明重组pro-UK的性质和天然pro-UK的性质相一致．     

Use of ethanol-eluted hydrophobic interaction chromatography in the purification of serum amyloid A.

A two-step procedure for the purification of the acute-phase reactant serum amyloid A from serum is described. A hydrophobic interaction chromatography medium, octyl-Sepharose CL4B, eluted with increasing concentrations of EtOH was used as the first step in the purification. The concentrate from this step was applied to a gel filtration column of Sephacryl S-200 and eluted with 10% formic acid. The overall recovery of purified serum amyloid A from serum was 56%. This represents the first time that serum amyloid A has been purified without the use of high concentrations of guanidine or urea. The method presented could easily be scaled up to allow the purification of large quantities of serum amyloid A or readily adapted to the purification of other serum apolipoproteins.     

A simple chromatographic procedure for the purification of the D dimer fragment from crosslinked fibrin

An improved procedure for the purification of fragment D dimer derived from crosslinked plasma fibrin is described which entails chromatofocusing chromatography using PBE 94 and polybuffer 74, and gel chromatography on Sephacryl S-300. The procedure provides a preparation of D dimer which behaves as a single macromolecular entity with molecular weight 190,000 in sedimentation equilibrium studies. Only a single protein band is observed in polyacrylamide gel electrophoresis conducted in the presence or absence of sodium dodecyl sulfate, while patterns characteristic of gamma'-gamma' chains are observed under denaturing conditions after reduction of the preparation with beta-mercaptoethanol. The D dimer contains no demonstrable E antigen by a range of electrophoretic and immunologic techniques. Advantages of this method for obtaining D dimer in high yield include the use of plasma as starting material, the use of a simple lysis regimen in the presence of Ca2+, and the use of simple chromatographic techniques performed under nondenaturing conditions.