限制性内切酶MspI的分离纯化

MspI内切酶是Ⅱ型限制内切酶。该酶首先是由R. J. Roberts从Moraxella species中提纯出来的,但有关该酶的纯化方法目前在国内外尚无报道。我们参照Roberts等人提取其它内切酶的方法,经过适当改进,从克隆MspI基因的大肠杆菌中分离、纯化了MspI。     

源于纤维单胞菌的聚阿拉伯糖内切酶的分离纯化

目的:分离纯化一种由纤维单胞菌属(Cellulomonose sp.)的细菌发酵产生的具有聚阿拉伯糖内切酶的性质的蛋白酶。方法:用薄板层析法(TLC)分离酶促反应的产物-寡糖,以Quanti-Scan软件计算薄板上条带的面积灰度值,以此定量,计算酶活力。用Bradford法测定蛋白含量。通过DEAE-Sepharose离子交换层析,SephacrylS-300分子筛和Blue-Dye活性染料亲和层析三种手段串联,对粗酶进行分离纯化,用SDS-PAGE测定纯度,SDS-PAGE和凝胶层析测定相对分子质量。结果:分离得到了电泳纯的阿拉伯糖内切酶,该酶的相对分子质量为45kD,蛋白酶比活性为15.42×10(3U/ug),纯化倍数为77.1。结论:该酶是一种阿拉伯糖内切酶,可以作为一种新型的工具酶,应用于结核分枝杆菌细胞壁的结构分析及寻找抗结核药物作用的靶点。     

限制性内切酶BamHI、EcoRI、PstI的分离纯化

限制性内切酶是核酸研究工作的重要工具酶,它的纯度直接影响基因分离,基因克隆以及序列分析等实验结果。为了获得品种繁多、纯度达到要求的酶,人们在分离纯化技术上作了不少努力。目前在分离限制性内切酶方法上以     

镰刀菌Q7-31T内切葡聚糖酶Egn21的分离纯化及酶学性质

【目的】为进一步研究镰刀菌Q7-31T产生的植物细胞壁降解酶的酶系信息。【方法】以1%(W/V)蛋白胨为氮源,0.5%(W/V)燕麦秸秆为碳源,20°C、120 r/min振荡培养3 d,诱导发酵培养菌株,获得的粗酶液经过Sephacry S-100凝胶柱层析和DEAE琼脂糖弱阴离子交换柱层析,最终得到纯化的内切葡聚糖酶,并对其进行酶学性质分析及串联质谱鉴定。【结果】研究表明:Egn21的分子质量为44.25 kDa,等电点为4.91;酶学特性研究显示:Egn21降解羧甲基纤维素的最适反应温度为40°C,在45°C以下比较稳定。该酶最适pH为6.0,在pH为5.0–8.0条件之间比较稳定。Co~(2+)、Zn~(2+)和Mg~(2+)对其没有明显作用,而Fe~(2+)、Ca~(2+)、K~(+)、Na~(+)和Mn~(2+)对酶活性有抑制作用,Hg~(2+)会使酶失去活性。【结论】从Q7-31T中分离纯化得到的内切葡聚糖酶Egn21,经过酶学特性与串联质谱鉴定结果显示其属于GH5家族。     

分离纯化限制性核酸内切酶的基本方法和纯度鉴定

为了适应研究工作需要,我们完成了BamHl、EcoRl、PstⅠ、AluⅠ、BglⅡ、SaiⅠ等十几种限制性核酸内切酶的分离纯化工作,样品在质量上达到国外同类产品的水平。这些酶制剂用于遗传工程及核酸结构功能研究已能取代相应的进口酶,促进了科研工作的发展。本文报道我们在制备限制性核酸内切酶中常用的几种方法,以及有实用意义的鉴定酶纯度的分析方法。一、材料与试剂 1.菌株:Arthrobacter luteus(ATCC21606),Bacillus globigii,Streptomyces albus G.     

小麦内切多聚半乳糖醛酸酶抑制蛋白的分离纯化研究

小麦内切多聚半乳糖醛酸酶抑制蛋白的分离纯化研究郑远旗,杨宗剑,李建吾,周立,吴文莲（四川大学生物系,成都６１００６４）余露（中国科学院成都生物研究所,成都６１００４１）关键词内切多聚半乳糖醛酸酶抑制蛋白;内切多聚半乳糖醛酸酶;纯化;小麦内切多聚半乳糖...     

Purification of the T4 endonuclease V

A new purification protocol has been developed for the rapid isolation to physical homogeneity of T4 endonuclease V. The enzyme was purified from an Escherichia coli strain which harbors a plasmid containing the T4 denV structural gene downstream of the lambda rightward promoter. The purification of the enzyme was monitored by pyrimidine dimer-specific nicking activity, Western blot analysis and silver or Coomassie Blue staining of SDS-polyacrylamide gels. Milligram quantities of the enzyme have been purified by the following procedure. After sonication of cells and removal of major cell debris, total protein and nucleic acids were passed over a single-stranded DNA agarose column. Endonuclease V was eluted at 650 mM KCl with a linear salt gradient yielding enzyme of approximately 20% purity and following dialysis, was applied to a chromatofocusing column. The enzyme elutes at pH 9.4 and is greater than 90% homogeneous at this step. The final purification step is CM-Sephadex chromatography which attains greater than 98% homogeneity.     

Dark Repair of Ultraviolet-Irradiated Deoxyribonucleic Acid by Bacteriophage T4: Purification and Characterization of a Dimer-Specific Phage-Induced Endonuclease

The purification and properties of an ultraviolet (UV) repair endonuclease are described. The enzyme is induced by infection of cells of Escherichia coli with phage T4 and is missing from extracts of cells infected with the UV-sensitive and excision-defective mutant T4V(1). The enzyme attacks UV-irradiated deoxyribonucleic acid (DNA) containing either hydroxymethylcytosine or cytosine, but does not affect native DNA. The specific substrate in UV-irradiated DNA appears to be pyrimidine dimer sites. The purified enzyme alone does not excise pyrimidine dimers from UV-irradiated DNA. However, dimer excision does occur in the presence of the purified endonuclease plus crude extract of cells infected with the mutant T4V(1).     

Endonuclease V of Escherichia coli.

A small endodeoxyribonuclease )2.3 S) that is active on single-stranded DNA has been extensively purified from Escherichia coli so as to be free of other known DNases. It has an alkaline pH optimum (9.5), requires Mg2+, and makes 3'-hydroxy and 5'-phosphate termini. The nuclease nicks duplex DNA, particularly if treated with OsO4, irradiated with ultraviolet light, or exposed to pH 5. The uracil-containing duplex DNA from the Bacillus subtilis phage PBS-2 is an especially good substrate; it is made acid-soluble by levels of the enzyme which fail to produce any acid-soluble material in other single-stranded or duplex DNAs. Neither RNA nor RNA-DNA hybrid are degraded by the enzyme. The enzyme specificity suggests that it might act at abnormal regions in DNA, so that its in vivo function could be to initiate an excision repair sequence. Its high activity on uracil-containing DNA could imply that the enzyme provides an alternative mechanism for excising uracil residues from DNA to the pathway utilizing uracil-DNA N-glycosidase. We suggest that this enzyme be designated as endonuclease V of E. coli.     

Hydration at the TD Damaged Site of DNA and its Role in the Formation of Complex with T4 Endonuclease V

Abstract

An analysis of the distribution of water around DNA surface focusing on the role of the distribution of water molecules in the proper recognition of damaged site by repair enzyme T4 Endonuclease V was performed. The native DNA dodecamer, dodecamer with the thymine dimer (TD) and complex of DNA and part of repair enzyme T4 Endonuclease V were examined throughout the 500 ps of molecular dynamics simulation. During simulation the number of water molecules close to the DNA atoms and the residence time were calculated. There is an increase in number of water molecules lying in the close vicinity to TD if compared with those lying close to two native thymines (TT). Densely populated area with water molecules around TD is one of the factors detected by enzyme during scanning process. The residence time was found higher for molecule of the complex and the six water molecules were found occupying the stabile positions between the TD and catalytic center close to atoms P, C3′ and N3. These molecules originate water mediated hydrogen bond network that contribute to the stability of complex required for the onset of repair process.     

Partial Suppression of Bacteriophage T4 Ligase Mutations by T4 Endonuclease II Deficiency: Role of Host Ligase

Endonuclease II-deficient, ligase-deficient double mutants of phage T4 induce considerably more deoxyribonucleic acid (DNA) synthesis after infection of Escherichia coli B than does the ligase-deficient single mutant. Furthermore, the double mutant can replicate 10 to 15% as well as wild-type T4, whereas the single mutant fails to replicate. When the E. coli host is also deficient in ligase, the double mutant resembles the single mutant. The results indicate that host ligase can substitute for phage ligase when the host DNA is not attacked by the phage-induced endonuclease II.     

Point Mutants in the D2a Region of Bacteriophage T4 Fail to Induce T4 Endonuclease IV

We have studied the properties of presumptive point mutants in the D2a region of bacteriophage T4. Dominance tests showed that the D2a mutation was recessive to the wild-type allele. The mutations were shown to map in the D2a region by complementation against rII deletions. The D2a mutations were also located between gene 52 and rIIB by two- and three-factor crosses. The mutants are located at at least two distinct loci in the D2a region. The point mutants grow normally on all hosts tested and none of the mutants makes T4 endonuclease IV. We propose the name "denB" for the D2a locus.     

Host DNA Degradation after Infection of Escherichia coli with Bacteriophage T4: Dependence of the Alternate Pathway of Degradation Which Occurs in the Absence of Both T4 Endonuclease II and Nuclear Disruption on T4 Endonuclease IV

Escherichia coli cells infected with T4 phage which are deficient in both nuclear disruption and endonuclease II exhibit a pathway of host DNA degradation which does not occur in cells infected with phage deficient only in endonuclease II. This alternate pathway of host DNA degradation requires T4 endonuclease IV.     

Purification and Some Properties of Plant Endonuclease from Scallion Bulbs

A plant endonuclease with 3′-nucleotidase activity was purified from scallion bulbs to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 38,000 by SDS-PAGE and 40,000 by Sephadex G-100 gel filtration. The enzyme rapidly hydrolyzed yeast RNA and denatured calf thymus DNA to acid-soluble substances, and hydrolyzed the plasmid pBR322 to yield small DNA fragments at low enzyme concentrations. These four activities were eliminated by treatments with EDTA and tetraethylenepentamine. The enzyme had maximum activity at pH 8.5-9.0 for 3′-AMP, 3′-GMP, and 3′-UMP, at pH 6.5 for 3′-CMP and yeast RNA, and at pH 6.0 for denatured calf thymus DNA and pBR322. During digestion of yeast RNA by the enzyme at pH 6.5, 5′-GMP was released most rapidly, followed by 5′-UMP, 5′-AMP, and 5′-CMP. These properties were different from those of endonucleases isolated from other sources such as mung bean sprouts and wheat seedlings.