Rhodococcus ruber CGMCC3090腈水合酶纯化、酶学性质及结晶研究

Rhodococcus ruber CGMCC309菌株为酰胺酶及腈水解酶双重缺陷菌株,研究表明该菌能产宽泛底物特异性的腈水合酶。对该菌株产生的新型腈水合酶(NHase-3090)进行纯化和结晶,并研究了其酶学性质。采用疏水、离子交换及凝胶过滤3种层析方法,使该酶纯化倍数达到17.14,得率高达26.2%。电泳分析表明,全酶分子量为105 kDa,由α(24.3 k Da)和β(28.0k Da)2个亚基组成,并构成α2β2四聚体。酶的最适p H和温度分别为7.5和30℃。该酶明显受不同金属离子影响。动力学研究表明,Km为178.8 m M;Vmax为209.1μmol/L·min·mg。研究发现3种金属离子Zn~(2+),CO~(2+)和Cd~(2+)有利于酶蛋白结晶。结晶最佳条件是:采用112-34#试剂(0.05mol/L水合硫酸镉、0.1mol/L HEPS和1.0mol/L三水醋酸钠),蛋白质浓度为15 mg/ml,结晶温度为16℃,p H为7.5,结晶时间为30 d。腈水合酶蛋白单晶经X射线衍射,分辨率达到了3.7。该腈水合酶的纯化和结晶为进一步深入研究其结构和功能奠定了基础。     

Purification,Characterization, and Crystallization of Crocodylus siamensis Hemoglobin

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P₅₀) and 1.96 (n value), and a small Bohr effect (δH⁺ = ?0.29) at a pH of 6.9–8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O₂ affinity at P₅₀ of 2.5 mmHg which may assure efficient utilization of the lung O₂ reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10–13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl₂·6H₂O] solution at a pH of 7.0–8.5.     

尿激酶催化结构域在毕氏酵母中的表达、纯化及结晶

利用重叠延伸PCR方法扩增尿激酶催化结构域的突变体基因片段,将其克隆至表达载体pPICZαA上,转化酵母X-33,用Zeocin筛选高拷贝数的酵母菌落.重组蛋白通过阳离子琼脂糖柱纯化,纯度达到99%,该仅含尿激酶催化结构域的突变体(C279A/N302Q),无需激活即具有尿激酶活性.用气相扩散法获得蛋白质晶体,其衍射分辨率达1.45!.     

辣椒疫霉PcCRN20-C蛋白的表达纯化及结晶 *

CRN(crinkling and necrosis-inducing protein)为疫霉菌在与寄主互作过程中分泌的一类特有胞质效应因子,干扰寄主细胞正常的生理代谢和功能。采用PCR法从辣椒疫霉LT1534菌株cDNA中克隆PcCRN20-C基因。该基因序列长783bp,编码261个氨基酸。构建重组表达载体,并转化大肠杆菌BL21(DE3)。在优化条件下诱导表达重组蛋白,利用Ni-NTA金属螯合层析、离子交换层析、分子筛层析和胰蛋白酶酶解技术获得高纯目的蛋白,SDS-PAGE分析表明,蛋白质分子量约为25kDa。采用座滴气相扩散法进行晶体制备和筛选,成功获得了蛋白质晶体,并通过X-射线衍射仪收集了晶体衍射花样。结合蛋白质晶体学方法,获得了有衍射的辣椒疫霉PcCRN20-C蛋白晶体,为进一步研究CRN蛋白的结构与病原菌致病机制提供参考资料。     

Salmonella typhimurium LT2 metF operator mutations

Summary Using an Escherichia coli lac deletion strain lysogenized with lambda phage carrying a metF-lacZ gene fusion (Flac), in which -galactosidase levels are dependent on metF gene expression, cis-acting mutations were isolated that affect regulation of the Salmonella typhimurium metF gene. The mutations were located in a region previously defined as the metF operator by its similarity to the E. coli metF operator sequence. Regulation of the metF gene was examined by measuring -galactosidase levels in E. coli strains lysogenized with the wild-type Flac phage and mutant Flac phage. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product, but not the vitamin B₁₂ control system mediated by the metH gene product. The results also demonstrate that negative control of the metF gene by the metH gene product and vitamin B₁₂ is dependent on a functional metJ gene product.Abbreviations Ap ampicillin - dNTP deoxyribonucleoside triphosphates - GM glucose minimal - Km kanamycin - L-agar Luria agar - LM lactose minimal - SAM s-adenosyl-L-methionine - TPEG phenylethyl -D-thiogalactoside - X-gal 5-bromo-4-chloro-3-indolyl -D-galactopyranoside - [] designates plasmid-carrier state - :: novel joint     

Plasmid-determined alterations of Salmonella typhimurium lipopolysaccharides

Summary Salmonella typhimurium Rc902 infected with derepressed ColIb mutants gave rise to changes in the composition of bacterial lipopolysaccharides (LPS). Bacteria carrying ColIbdrd7, derepressed in transfer, exhibited a marked decrease in the content of all 0-side-chain sugars of LPS. Similar effects were found upon the introduction of R64-11, also derepressed in transfer. In LPS of S. typhimurium containing ColIbdrd2, derepressed in colicin synthesis, a decrease of abequose content associated with an increase of glucose level was observed. Bacteria carrying the wild-type ColIb, the revertant of a drd mutant to the wild type, or the non colicinogenic strain resulting from the elimination of ColIbdrd2, showed no changes in the sugar composition of LPS.     

华根霉胞内脂肪酶同功酶酶学性质研究

华根霉(Rhizopus chinensis CCTCCM201021)细胞破碎液经硫酸铵分级盐析、Phenyl-Sepharose FF疏水层析、DEAE-Sepharose FF离子交换层析和Sephadex G100凝胶层析得到两种脂肪酶同功酶:Lip1和Lip2。SDS-PAGE显示其亚基分子量分别为59.2kD和39.4kD。Lip1和Lip2的最适反应pH和最适反应温度相近,分别为8.0、8.5和40℃、35℃。但底物专一性差异明显:Lip1对pNP脂肪酸酯的长链脂肪酸有较高的专一性,Lip2对pNP脂肪酸酯的短链脂肪酸专一性较好;Lip1对三油酸甘油酯表现1,3-位置特异性,而Lip2没有位置选择性。1mmol/L的Ca2 、Mg2 对Lip1、Lip2有较好的激活作用;SDS强烈抑制酶活力。Lip1、Lip2在环己烷、正己烷、正庚烷和异辛烷(30%V/V)中稳定性良好。     

Functional homology of fla genes between Salmonella typhimurium and Escherichia coli

Summary Genetic studies have shown the presence of more than 20 fla genes indispensable for the formation of flagella in Salmonella typhimurium and Escherichia coli. Functional homology of the fla genes in these two bacterial species was examined through intergeneric complementation tests by bacteriophage Pl-mediated transduction from E. coli donors to S. typhimurium recipients. It was found that most of the fla gene products in these two bacterial species were interchangeable and the following correspondence was established (S. typhimurium genes vs. E. coli genes): flaFIV to flaV; flaFV to flaK; flaFVII to flaL; flaFIX to flaM; flaC to flaH; flaM to flaG; flaE to flaI; flaAI to flaN; flaAII·1 to flaB; flaAIII to flaC; flaS to flaO; flaR to flaE; flaQ to flaA; and flaB to flaR. These results suggest that the chromosomal alignment of the functionally homologous genes is very similar in these two bacterial species. Furthermore, five additional fla genes were inferred to exist in E. coli in addition to the fla genes already identified. They were termed flaU, flaX, flaY, flaZ, and flbB (flb is equivalent to fla), which corresponded to flaFI, flaFVI, flaFVIII, flaFX, and flaK of Salmonella in this order. The flaK mutants of E. coli showed no complementation with any of the flaFV, flaFVI, flaFVII, flaFVIII, or flaFIX mutants of Salmonella.     

来自于变性链球菌的脱氧胞嘧啶核苷酸脱氨酶的表达，纯化，结晶以及初步晶体学研究

脱氧胞嘧啶核苷酸脱氨酶属于脱氧胞苷酸脱氨家族.对来自于变性链球菌UA159的脱氧胞嘧啶核苷酸脱氨酶进行了克隆,在大肠杆菌中进行了表达,最后纯化.快速液相分子排阻色谱分析表明这种酶在溶液中形成六聚体.利用悬滴气相扩散技术获得了这个蛋白的晶体.在北京同步辐射的3W1A线站,收集了衍射分辨率到达3.1!的数据.这个晶体属于P213空间群,其晶胞参数为a=b=c=113.2",!="=#=90°.计算可得马修斯系数为3.6#3·Da-1,据此可估计在一个不对称单位中含有两个单亚基.据目前所知,这是第一个关于野生型的脱氧胞嘧啶核苷酸脱氨酶的结晶学报道.     

Purification,Characterization, and Crystallization of Two Types of Lipase from Rhizopus niveus

The purification and some properties of two types of lipase (Lipase I and Lipase II) from Rhizopus niveus are described. The enzymes were purified to homogeneity by column chromatographies on DEAE-Toyopearl (1 pass) and CM-Toyopearl (2 passes). Lipase I consists of two polypeptide chains [a small peptide with sugar moiety (A-chain) and a large peptide of molecular weight 34,000 (B-chain)]. Lipase II has a molecular weight of 30,000 consisting of a single polypeptide chain. Lipase I appeared to be converted to Lipase II by limited proteolysis by a specific protease a small amount of which is in the culture supernatant from Rh. niveus, because one of the peptides formed has the same N-terminal sequence and C-terminal amino acid as Lipase II, as well as the molecular mass estimated by SDS-PAGE. Lipase I had a pH optimum of 6.0–6.5 and a temperature optimum of 35°C, while, for Lipase II these values were pH 6.0 and 40°C. Both enzymes were obtained in the crystalline state using the hanging drop method of vapor diffusion and PEG as the precipitating agents.     

Induction and cleavage of Salmonella typhimurium UmuD protein

Summary Two different chromosomal locations of major genes controlling extreme resistance to potato virus X (PVX) were found by restriction fragment length polymorphism (RFLP) analysis of two populations segregating for the resistance. The resistance geneRx1 mapped to the distal end of chromosome XII, whereasRx2 was located at an intermediate position on linkage group V in a region where reduced recombination and segregation distortion have also been observed. These linkage anomalies were due to abnormal behaviour of the chromosome contributed by the resistant parent P34. The results presented were obtained using two different strategies for mapping genes of unknown location. One approach was the use of probes revealing polymorphic loci spread throughout the genome and resulted in the mapping ofRx1. The second approach was based on the assumption of possible linkage between the resistance gene and clone-specific DNA fragments introduced from a wild potato species.Rx2 was mapped by adopting this strategy.     

Purification,Characterization, and Crystallization of Monoamine Oxidase from Escherichia coli K-12

The gene for monoamine oxidase (MAO) was cloned from an Escherichia coli genomic library and MAO was overproduced in the periplasmic space. The enzyme was purified to homogeneity by preparation of a periplasmic fraction, followed by ammonium sulfate fractionation and DEAE-cellulose column chromatography. Crystals were obtained by the hanging drop method using sodium citrate as a precipitant. The enzyme was found to be a dimer of identical subunits with a molecular weight of 80,000, and showed the highest activity at pH 7.5 and 45°C. The enzyme was inhibited by a MAO specific inhibitor, hydroxylamine, hydrazine, phenelzine, isoniazid, and tranycypromine. The enzyme oxidized tyramine, phenethylamine, and tryptamine at higher rates, but not oxidized diamine and polyamines such as putrecine and spermine. The antibody against E. coli MAO cross-reacted with purified MAO A from Klebsiella aerogenes.     

鼠伤寒沙门氏菌baeR过表达株的构建及其耐药性

[背景]鼠伤寒沙门氏菌（Salmonella typhimurium）是一种重要的人兽共患病原菌,其多重耐药性问题日益严重,双组分系统可调控鼠伤寒沙门氏菌的耐药性。[目的]通过构建鼠伤寒沙门氏菌baeR过表达株及回补株探究BaeSR双组分系统对鼠伤寒沙门氏菌耐药性的影响。[方法]在BaeSR双组分系统和AcrB外排泵双缺失株（CRΔbaeSRΔacrB）的基础上构建baeR过表达株（CRpbaeRΔbaeSRΔacrB）及baeR回补株（CRcbaeRΔbaeSRΔacrB）,测定双缺失株、回补株和过表达株的最小抑菌浓度（minimum inhibitory concentration,MIC）,并对其生长特性、生物膜形成能力及运动性进行分析。采用转录组学技术筛选与耐药相关的差异表达基因,RT-qPCR验证耐药相关基因。[结果]构建了鼠伤寒沙门氏菌baeR过表达株和baeR回补株。与双缺失株相比,过表达株对氧氟沙星、恩诺沙星、氟苯尼考、乙酰甲喹、头孢他啶、头孢噻呋、阿莫西林和氨苄西林的MIC分别升高2-256倍,对大观霉素、安普霉素的MIC下降了50%;与双缺失株相比,回补株对头孢他啶...     

Salmonella typhimurium mutants affecting establishment of lysogeny

Summary Salmonella typhimurium mutants have been isolated in which phage P22 fails to establish lysogeny. These appear to be defective in cAMP metabolism. A phage mutation overcoming the bacterial defect has been mapped between gene c ₁ and gene 12.     

High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation

Summary Salmonella typhimurium and S. typhi were transformd with high efficiency by electroporation. Transformation efficiencies of up to 10¹⁰ transformants per g of pBR322 were obtained. In contrast to chemical transformation methods, neither the smooth lipopolysaccharide of S. typhimurium nor the Vi capsular polysaccharide of S. typhi greatly affected transformation efficiency. The introduction of a galE mutation slightly improved transformation efficiency in S. typhimurium (< tenfold) while the Vi antigen of S. typhi had no detectable effect. The transformation efficiency of S. typhimurium with DNA derived from Escherichia coli was increased greatly by the removal of the hsd restriction system (100-fold). Under these conditions electroporation can be used for the routine and direct transformation of Salmonella strains with partially purified (alkaline lysis) plasmid DNA from E. coli.     

鼠羧肽酶原B在毕赤酵母中的表达、纯化与鉴定

为了在毕赤酵母中表达鼠羧肽酶原B(procarboxypeptidaseB,proCPB)蛋白,以RT-PCR法从SD鼠胰腺细胞中克隆了proCPB基因,将其插入pPIC9载体,PEG1000介导转入毕赤酵母GS115细胞,在甲醇的诱导下,实现了proCPB在毕赤酵母中的成功表达。通过发酵条件的优化,使用BMGY(pH6·0)培养基,添加0·5%的酪蛋白水解物,于28℃,在起始OD600达10·0时,每隔12h补加0·5%的甲醇,重组酵母GS115-proCPB表达的产物量可达到最高(500mg/L),表达时间可达120h,表达的目的蛋白占总蛋白的94%以上。通过纯化条件的优化,采用两步疏水层析,可使目的蛋白的纯度达96%以上,蛋白得率达38%,重组proCPB活化后所得CPB的比活力可达110u/mg(CPB标准品为180u/mg)。相对分子量测定表明重组蛋白的分子量与理论值极相近,N-端氨基酸测序进一步表明proCPB基因在毕赤酵母中得到了正确的表达和翻译后加工修饰。     

西伯利亚鲟卵黄脂磷蛋白的分离纯化及性质

卵黄是鱼类胚胎发生期的主要营养物质,卵黄的含量和质量对于早期幼体维持生命和生长发育至关重要.本研究采用Sephacryl S-300凝胶过滤层析法和蛋白质电泳技术分离纯化西伯利亚鲟(Acipenser baerii)卵黄脂磷蛋白(lipovitellin,Lv),层析洗脱共得到7个蛋白峰.对每个峰进行SDS-PAGE电泳及油红O、甲基绿和Schiff试剂特异染色,峰b蛋白均呈阳性,表明峰b蛋白为西伯利亚鲟卵中的一种卵黄脂磷蛋白,SDS-PAGE电泳分析表明,其由3个亚基构成,相对分子质量分别为30.6 ku、40.8 ku和76.7 ku.对西伯利亚鲟Lv氨基酸组成进行分析,证明是一种含有相对较多天冬氨酸、赖氨酸、谷氨酸、丝氨酸、缬氨酸和亮氨酸的蛋白,并且所含鲜味氨基酸含量比其他鱼偏高.     

envB mutations confer UV-sensitivity to Salmonella typhimurium and UV-resistance to Escherichia coli

Summary An envB mutation isolated in Salmonella typhimurium LT2 was transferred by conjugation to Escherichia coli K-12. The mutation produced the same alterations in E. coli as in S. typhimurium concerning cell shape, sensitivity to drugs, autolysis, and fermentation of carbohydrates. However, although the mutation conferred sensitivity to UV irradiation in Salmonella, in E. coli it behaved as a genuine envB mutation producing resistance to UV inactivation. The fact that the mutation produced opposite effects in the survival of UV-irradiated S. typhimurium and E. coli discloses an intriguing difference between these closely related species.Career Investigator of the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina     

Purification and some properties of cytosine deaminase from Salmonella typhimurium

Cytosine deaminase (EC 3.5.4.1) from Salmonella typhimurium has been purified 419-fold to apparent homogeneity. SDS polyacrylamide gel electrophoresis indicated that the final cytosine deaminase preparation was homogenous. The molecular weight of cytosine deaminase was determined to be approx. 230 000 containing four identical subunits with each subunit having a molecular weight of 54 000. Cytosine deaminase has a pH optimum of 7.30 to 7.50 and a temperature optimum of 45 to 50°C. Cytosine was deaminated specifically; 5-fluorocytosine was deaminated to a lesser extent. The K_m and V values for cytosine were 0.74 mM and 47.16 μmole/min, respectively. As effectors of enzyme activity, PP_i stimulated the deamination while metal ions and orotidine monophosphate inhibited it. The physical characteristics of cytosine deaminase lend credence to its proposed salvage role in pyrimidine metabolism as indicated previously by physiological studies (West, T.P. and O'Donovan, G.A., J. Bacteriol. (1982) 149, 1171–1174).