非盐依赖层析的研究与应用

在非盐依赖层析操作中,吸附介质的成分、结构、密度等性质得到改进,所以料液离子强度的变化不会明显影响吸附 . 与常用的离子交换层析、疏水层析、亲硫层析等方法相比,该类技术能够降低对料液预处理的要求,提高蛋白质的稳定性,同时简化层析操作、降低纯化成本,具有大规模分离纯化蛋白质的潜力 . 近年来开发的多种非盐依赖层析介质与方法,都已在蛋白质纯化中得到应用 .     

一种改进的烟草多酚氧化酶Ⅱ纯化方法

目的:选择不同的分离、纯化步骤并比对分析,筛选出纯化烟草中多酚氧化酶(PPO)的优化组合方案。方法:采用分段盐析、DEAE-SepharoseFastflow和SephadexG-150柱层析纯化PPO,通过测定和比较酶活性筛选最佳条件。结果:确定了最佳盐析浓度(40%)和柱层析条件,SDS-PAGE、FPLC以及动力学常数的检测结果表明,纯化出的蛋白质相对分子质量为42000,Km为1.2mmol/L,得到了纯化91倍的烟草多酚氧化酶Ⅱ。结论:优化方案减少了有机溶剂分级沉淀、阳离子交换层析等步骤,使纯化过程大大缩短。     

灵芝免疫调节蛋白LZ-8的制备和晶体分析

LZ-8蛋白是从灵芝菌丝中分离到的真菌免疫调节蛋白,具有多种免疫调节功能,然而这一蛋白的作用机制尚不清楚。通过蛋白质晶体结构的解析,能够得到蛋白质空间结构特点,从而阐述蛋白质功能的机制。旨在得到LZ-8蛋白的晶体,并获得空间结构数据。以pET21a为表达载体,获得诱导表达的rLZ-8,通过亲和层析、分子筛凝胶层析和阴离子交换层析纯化,蛋白纯度在98%以上,采用悬滴气相扩散法得到蛋白晶体,并获得3.2?数据,为进一步对真菌免疫调节蛋白功能和结构的研究奠定了基础。     

蛋白质分离纯化方法的研究进展

蛋白质作为生命物质基础之一,存在于自然界中复杂的混合体系中。蛋白质在细胞中的含量极低,将其从复杂体系中分离出来并保持其活性,难度较大,因此分离纯化蛋白质的方法受到生命科学领域广泛关注。本文首先阐述了蛋白质的预处理,其次阐述了蛋白质分离纯化的各种方法如沉淀、层析、离心等,重点阐述了蛋白质新纯化的各种原理,以及每种方法的适用范围。有助大家更全面、更彻底的了解蛋白纯化技术的发展,以期为今后开展蛋白质的制备及应用提供一定理论依据和实验指导。     

乳腺生物反应器表达的重组人乳清白蛋白的高效分离纯化

利用乳腺生物反应器高效地表达重组人乳清白蛋白,但是目标产物分离纯化的难度较大。通过分子模拟计算比较待分离原料中主要蛋白组分的物化性质,包括表面电势和表面疏水性,在此基础上设计了高分辨率、快速的分离纯化工艺。通过硫酸铵沉淀的正交试验条件优化,有效地去除了干扰层析精制过程的IgG杂质,提高了后续疏水层析的稳定性,从而成功地分离开目标蛋白及与其同源的牛乳清白蛋白杂质,得到纯度>95%的rHLA纯品,工艺回收率48.6%。乳糖合成活性和圆二色谱检测结果表明,纯化rHLA具有调节β-1,4-半乳糖苷转移酶活性和天然的空间结构。     

蜗牛酶中一种人参皂苷Rb1水解酶的分离纯化

通过DEAE-Sepharose离子交换分段层析,DEAE-Sepharose离子交换梯度层析和SephadexG-100凝胶过滤层析三种方法的联用从中华白玉蜗牛消化酶中分离出一种人参皂苷Rb1水解酶。分离后该酶在SDS-PAGE上呈单一蛋白质务带。应用SDS-PAGE和凝胶过滤层析对分子量的测定,提示该酶是由4个分子量为110～115kD的相同亚基组成的同源四聚体。Rb1为底物的动力学参数Km和Vmax分别为0．790mmol／L和10．192μmol／min／mg。该酶对人参皂苷Rb1糖键进行有选择的水解,可水解人参皂苷Rb1C50位的一个糖苷键生成人参皂苷Rd。     

虎血清免疫球蛋白（IgG）的纯化及纯度、活性鉴定

目的 建立高纯度、高活性的虎血清IgG纯化方法。方法 用饱和硫酸铵沉淀虎血清得到IgG粗品;结合Hitrap Protein A亲和层析预装柱及阴离子交换层析法对粗品IgG进一步分离纯化,采用PAGE电泳和Western-Blot免疫印迹法鉴定IgG纯度和免疫活性。结果 80 mL虎血清亲和纯化得到84 mg IgG,阴离子交换层析纯化得到30 mg虎的IgG纯品。结论 建立了简便快速、纯度高、活性好的虎血清IgG的分离纯化方法,为虎血清IgG二级抗体的制备提供了高纯度、活性好的一级抗体免疫原。     

沙雷氏菌(Serratia marcescens)Yj1的分离鉴定及菌体有机磷降解酶的分离纯化

从大豆土壤中分离纯化得到一株具有卵磷脂和乐果降解能力的菌株Yj1,对该菌株进行鉴定、生长条件优化、酶活性鉴定以及有机磷降解酶的分离纯化。结果表明,Yj1与Serratia marcescens WW4(CP003959.1)的16S r DNA相似度为99%。正交试验对所需培养基进行优化,得到该菌株的最佳生长条件为甘露糖、蛋白胨和p H 8的组合。Yj1菌株在两种磷源条件下,菌株生长量均很低,但72 h内以大豆卵磷脂为磷源时的菌体生长情况优于乐果。以大豆卵磷脂为磷源时酸性磷酸酶、碱性磷酸酶与有机磷降解酶活性明显高于以乐果为磷源时的酶活,且72 h内碱性磷酸酶活性一直都高于酸性磷酸酶和有机磷降解酶。硫酸铵沉淀法结合阳离子交换层析成功从Yj1菌体中分离纯化了有机磷降解酶,SDS-PAGE结果显示纯化的蛋白为单一条带。且阳离子交换层析的提纯倍数是硫酸氨沉淀的5.303倍,硫酸氨沉淀为粗酶的1.416倍。     

姜老师信箱

蛋白质研讨班学员问:姜老师,您好!我在蛋白质分离纯化方面有两个问题想向您请教:1、工厂规模化生产蛋白(分子量约为15kDa),除了采用各种层析方式外,一般还可以用什么方法来对蛋白质进行浓缩?与实验室规模的蛋白纯化相比是否有不同之处?     

HeLa细胞端粒酶的分离纯化及其蛋白质组分的研究

根据端粒酶含有蛋白质组分和RNA组分的特点,采用寡核苷酸亲和纯化法从HeLa细胞蛋白粗提物中分离纯化人类端粒酶,纯化产物以TRAP法检测其延伸端粒活性,并采用RNA印迹法进行鉴定,然后从纯化产物中分离蛋白质组分,以SDS-聚丙烯酰胺凝胶电泳检测其蛋白质亚基成分,可见到4种蛋白质亚基成分,与蛋白质分子质量标准比较,有两条位置接近212.2 ku,一条接近116.0 ku,一条接近42.7 ku.结果表明,蛋白质寡核苷酸亲和纯化法一步性分离纯化HeLa细胞端粒酶可得到端粒酶活性片段.     

Isolation and characterisation of chitosanase from Bacillus sp. 739 strain

The specific nature of the chitosanase activity of the strain Bacillus sp. 739 has been determined. Maximum enzyme activity was observed in a medium containing the biomass of the fruiting bodies of the fungus Macrolepiota procera. The chitosanase was purified to homogeneity using chromatography on DEAE-Sephadex A-50 and Toyopearl HW-50. The molecular weight of the enzyme, assessed by electrophoresis (the Laemmli procedure) approximated 46 kDa. Temperature and pH optima of the purified chitosanase were in the ranges 45-55 degrees C and 6.0-6.5, respectively. Time to half-maximum inactivation of the enzyme at 50 degrees C was equal to 1 h. With colloidal chitosan as the substrate, the value of K(M) of the purified chitosanase was equal to 25 mg/ml. The enzyme also exhibited a weak ability to hydrolyze colloidal chitin.     

来自拟青霉属真菌的壳聚糖酶的分离纯化、理化性质及降解产物的分析(英文)

从来自拟青霉属真菌Paecilomyces sp.CS-Z的发酵液中获得一种壳聚糖酶,该酶被纯化了9.4倍,产率为48.2%。经SDS-PAGE分析确定为单一条带,分子量为29kDa,其最适pH为6.0–6.5,最适温度为55℃,在80℃处理60min后,能保持较好的热稳定性,Hg2+完全抑制了酶活,对脱乙酰度85%–95%的壳聚糖具有较高的水解活性,而对几丁质和羧甲基纤维素无活性。薄层层析和质谱分析表明该酶是一种内切酶,其水解产物为聚合度大于6的壳寡糖,其理化性质与至今报道的壳聚糖酶有所不同,为壳聚糖酶的开发提供了重要的实验依据。     

Purification and characterization of West Nile virus nucleoside triphosphatase (NTPase)/helicase: evidence for dissociation of the NTPase and helicase activities of the enzyme

The nucleoside triphosphatase (NTPase)/helicase associated with nonstructural protein 3 of West Nile (WN) virus was purified from cell culture medium harvested from virus-infected Vero cells. The purification procedure included sequential chromatography on Superdex-200 and Reactive Red 120 columns, followed by a concentration step on an Ultrogel hydroxyapatite column. The nature of the purified protein was confirmed by immunoblot analysis using a WN virus-positive antiserum, determination of its NH(2) terminus by microsequencing, and a binding assay with 5'-[(14)C]fluorosulfonylbenzoyladenosine. Under optimized reaction conditions the enzyme catalyzed the hydrolysis of ATP and the unwinding of the DNA duplex with k(cat) values of 133 and 5.5 x 10(-3) s(-1), respectively. Characterization of the NTPase activity of the WN virus enzyme revealed that optimum conditions with respect to the Mg(2+) requirement and the monovalent salt or polynucleotide response differed from those of other flavivirus NTPases. Initial kinetic studies demonstrated that the inhibition (or activation) of ATPase activity by ribavirin-5'-triphosphate is not directly related to changes in the helicase activity of the enzyme. Further analysis using guanine and O(6)-benzoylguanine derivatives revealed that the ATPase activity of WN virus NTPase/helicase may be modulated, i.e., increased or reduced, with no effect on the helicase activity of the enzyme. On the other hand the helicase activity could be modulated without changing the ATPase activity. Our observations show that the number of ATP hydrolysis events per unwinding cycle is not a constant value.     

腐皮镰孢菌壳聚糖酶的酶学性质研究及其在酿酒酵母工业菌株中的表达

摘要：【目的】本研究旨在了解腐皮镰孢菌（Fusarium solani）壳聚糖酶的基本酶学性质及其在壳寡糖生产中的应用,构建能高效分泌表达壳聚糖酶的酿酒酵母工业菌株。【方法】采用RT-PCR扩增腐皮镰孢菌壳聚糖酶的cDNA序列;通过组氨酸标签,纯化得到E. coli表达的重组壳聚糖酶,并进行基本酶学性质研究;以薄层层析、高效液相色谱等技术对该酶的酶解产物进行分析;通过马克斯克鲁维酵母（Kluyveromyces marxianus）菊粉酶信号肽（INU1A）实现壳聚糖酶在酿酒酵母工业菌株N-27中的分泌表     

球孢白僵菌胞外壳聚糖酶的纯化和性质*

球孢白僵菌Beauveria bassiana 1316-V1的培养上清液经硫酸铵分级沉淀,Sephadex G-75凝胶过滤,Chitosan-bead亲和层析,第二次Sephadex G-75凝胶过滤, 得到电泳纯的一种胞外壳聚糖酶,比活力达到45u/mg 。此酶的分子量为36 kD; 最适酶反应温度为60℃;最适pH为4.0;最适离子强度为 0.25mol/L NaCl; 37℃以下,pH 2.0~5.0之间稳定性好; Cu2+、Hg2+、Pb2+、Ni2+ 对该酶有强烈抑制作用;Ag+、Mn2+也有较强抑制作用;Fe2+有轻微激活作用。该壳聚糖酶是一种糖蛋白,含糖约为12.6%。酶的最适底物为脱乙酰度为90%的胶体壳聚糖;也能轻微水解CMC、DEAE-Cellulose和胶体几丁质;但不能水解片状的壳聚糖和几丁质。     

Isolation and Characterization of Chitosanase from the Strain Bacillus sp. 739

The specific nature of the chitosanase activity of the strain Bacillus sp. 739 was determined. Maximum enzyme activity was observed in a medium containing biomass of the fruiting bodies of the fungus Macrolepiota procera. The chitosanase was purified to homogeneity by chromatography on DEAE-Sephadex A-50 and Toyopearl HW-50. The molecular weight of the enzyme assessed by electrophoresis (the Laemmli procedure) approximated 46 kDa. The temperature and pH optima of the purified chitosanase were in the ranges 45–55°C and 6.0–6.5, respectively. Time to half-maximum inactivation of the enzyme at 50°C was equal to 1 h. With colloidal chitosan as the substrate, the value of K of the purified chitosanase was equal to 25 mg/ml. The enzyme also exhibited a weak ability to hydrolyze colloidal chitin.     

Purification, characterization, and gene analysis of cellulase (Cel8A) from Lysobacter sp. IB-9374

An enzyme that has both beta-1,4-glucanase and chitosanase activities was found in the culture medium of the soil bacterium Lysobacter sp. IB-9374, a high lysyl endopeptidase-producing strain. The enzyme was purified to homogeneity from the culture filtrate using five purification steps and designated Cel8A. The purified Cel8A had a molecular mass of 41 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A pH optimum of 5.0 was found for the beta-1,4-glucanase activity, and pH optima of 5.0 and 7.0 were found for the chitosanase activity. Nucleotide sequencing of the Cel8A gene yielded a deduced amino acid sequence that comprises a 33-amino acid, N-terminal signal peptide and a mature enzyme consisting of a 381-residue polypeptide with a predicted molecular mass of 41,241 Da. The amino acid sequence of the Cel8A, which contains the catalytic module of glycosyl hydrolase family 8, is homologous to beta-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.     

Purification and characterization of a phosphatidylinositol 4-kinase from bovine uteri

Phosphatidylinositol 4-kinase has been purified 10,148-fold to a specific activity of 2.7 mumol/mg/min from bovine uteri. This purification was accomplished by detergent extraction of an acetone powder, ammonium sulfate precipitation, and chromatography on MonoQ, S-Sepharose, MonoP, and hydroxylapatite columns. The purified enzyme has a molecular mass of 55 kDa and appears to be monomeric. Kinetic analyses of the enzymatic activity demonstrated apparent Km values of 18 microM and 22 micrograms/ml (approximately 26 microM) for ATP and phosphatidylinositol, respectively, optimal activity in the pH range of 6.0-7.0, and a sigmoidal dependence of enzymatic activity on [Mg2+]. Ca2+ inhibited the enzyme at nonphysiological concentrations with 50% inhibition observed at a free [Ca2+] of approximately 300 microM. The purified enzyme efficiently utilized both ATP and 2'-deoxy-ATP as phosphoryl donors and specifically phosphorylated phosphatidylinositol on the fourth position. No phosphatidylinositol-4-phosphate 5-kinase activity was observed in the purified enzyme preparations. To our knowledge, this is the first reported purification of a phosphatidylinositol-specific phosphatidylinositol 4-kinase.     

Purification and Characterization of d-Aminoacylase from Alcaligenes faecalis DA1

A d-aminoacylase from Alcaligenes faecalis DA1 has been purified to homogeneity by a simple purification procedure with two columns, Fractogel DEAE-650 and HW-50. The specific activity of the purified enzyme was found to be 580 U/mg of protein with N-acetyl-dl-methionine as the reaction substrate. The apparent molecular weight and isoelectric point of this enzyme were determined to be 55,000 and 5.4, respectively.     

Molecular characterization of a novel family-46 chitosanase from Pseudomonas sp. A-01

Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5-8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.