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Intracellular localization of P1 ParB protein depends on ParA and parS

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1999年第26期96卷 14905-14910页

作者： Erdmann, N Petroff, T Funnell, BE Univ Toronto Dept Mol & Med Genet Toronto ON M5S 1A8 Canada

The P1 partition system promotes faithful plasmid segregation during the Escherichia coli cell cycle. This system consists of two proteins, ParA and ParB, that act on a plasmid site called parS, By immunofluorescence microscopy, we observed that ParB localizes to discrete foci that are most often located close to the one-quarter and three-quarters positions of cell length, The visualization of ParB foci depended completely on the presence of parS, although their visualization was independent of the chromosomal context of pars (in P1 or the bacterial chromosome). In integration host factor-defective mutants, in which ParB binding to parS is weakened, only a fraction of the total pool of ParB had converged into foci, Taken together, these results indicate that pars recruits a pool of ParB into foci and that the resulting ParB-parS complexes serve as substrates for the segregation reaction. In the absence of ParA, the position of ParB foci in cells is perturbed, indicating that at least one of the roles of ParA is to direct ParB-parS complexes to the proper one-quarter positions from a cell pole. Finally, inhibition of cell division did not inhibit localization of ParB foci in cells, indicating that the positioning signals in the E. coli host that are needed for P1 partition do not depend on early division events.

关键词：细菌蛋白质类/分离和提纯细菌噬菌体P1/遗传学细胞区室化细胞周期大肠杆菌/细胞学大肠杆菌/遗传学荧光抗体技术整合宿主因子类质粒/遗传学原病毒/遗传学

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Crystal engineering:: deletion mutagenesis of the 24 kDa fragment of the DNA gyrase B subunit from Staphylococcus aureus

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ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 1999年第9期55卷 1626-1629页

作者： Dale, GE Kostrewa, D Gsell, B Stieger, M D'Arcy, A F Hoffman La Roche Ltd Pharma Preclin Res CH-4070 Basel Switzerland

The 24 kDa fragment of DNA gyrase B from Staphylococcus aureus was expressed in Escherichia coli and purified for crystallization. Crystals of the wild-type protein grew in the presence of cyclothialidine but proved difficult to reproduce. In order to improve the crystallization, the flexible regions of the protein were deleted by mutagenesis. The mutant proteins were analyzed by differential scanning calorimetry and the most stable mutants produced crystals. It was possible to reproducibly grow in, the microbatch system single well defined crystals which belonged to the space group C2 and diffracted isotropically to approximately 2 Angstrom resolution.

关键词：细菌蛋白质类/生物合成细菌蛋白质类/化学细菌蛋白质类/遗传学细菌蛋白质类/分离和提纯结晶结晶学 X线 DNA促旋酶 DNA拓扑异构酶类 Ⅱ型/生物合成 DNA拓扑异构酶类 Ⅱ型/化学 DNA拓扑异构酶类 Ⅱ型/遗传学 DNA拓扑异构酶类 Ⅱ型/分离和提纯大肠杆菌/酶学大肠杆菌/遗传学基因缺失诱变肽碎片/生物合成肽碎片/化学肽碎片/遗传学肽碎片/分离和提纯金黄色葡萄球菌/酶学

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IDENTIFICATION OF UUG CODON AS A TRANSLATIONAL INITIATION CODON INVIVO

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JOURNAL OF MOLECULAR BIOLOGY 1975年第2期95卷 327-330页

作者： FILES, JG WEBER, K COULONDRE, C MILLER, JH HARVARD UNIV BIOL LABS CAMBRIDGE MA 02138 USA MAX PLANCK INST BIOPHYS CHEM DEPT BIOCHEM GOTTINGEN FED REP GER UNIV GENEVA DEPT MOLEC BIOL GENEVA SWITZERLAND

The lac repressor protein was purified from an Escherichia coli strain carrying an amber mutation in the lacI gene and the tyrosine-inserting amber suppressor, Su3. Protein sequencing showed a change at position 62 in the repressor polypeptide chain from leucine to tyrosine, proving that the amber was derived from a UUG codon at this point in the message. This establishes UUG as an initiation codon in vivo , since it has been previously shown that translational reinitiation can occur at position 62.

关键词：氨基酸序列细菌蛋白质类/分析细菌蛋白质类/分离和提纯密码子大肠杆菌/分析基因遗传学微生物突变肽链起始翻译蛋白质生物合成 RNA 信使

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Purification of His-tagged proteins by immobilized chelate affinity chromatography: The benefits from the use of organic solvent

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PROTEIN EXPRESSION AND PURIFICATION 2000年第1期18卷 95-99页

作者： Franken, KLMC Hiemstra, HS van Meijgaarden, KE Subronto, Y den Hartigh, J Ottenhoff, THM Drijfhout, JW Leiden Univ Med Ctr Dept Immunohematol & Blood Bank NL-2300 RC Leiden Netherlands Leiden Univ Med Ctr Dept Clin Pharmacol NL-2300 RC Leiden Netherlands

Recombinant proteins overexpressed in and purified from Escherichia coli contain impurities that are toxic in biological assays. The application of affinity purification procedures is often not sufficient to remove these toxic components, We here describe a simple and fast, one-step protocol to remove these impurities highly efficiently. Four recombinant proteins were overexpressed in E, coli as His-tagged fusion proteins and purified by immobilized metal chelate affinity chromatography on Ni-NTA beads. Depending on the protein, the composition of the lysis buffer, and the washing protocol, various impurities appeared to be present in the purified protein preparations. Here we show how the use of 60% isopropanol during washing steps removed most of these contaminants from the end products, In addition to the removal of proteins that aspecifically adhere to the beads or to the tagged protein, this procedure was particularly useful in removing endotoxins, Moreover, we show that detergents such as NP-40, that are necessarily employed during lysis, are also efficiently removed, Finally, we show that proteins are able to refold correctly after isopropanol treatment. Thus, the resulting end products contain significantly less contaminating E. coli proteins, endotoxins, and detergents. (C) 2000 Academic Press.

关键词： 2-丙醇细菌蛋白质类/化学细菌蛋白质类/遗传学细菌蛋白质类/分离和提纯色谱法亲和/方法洗涤剂/分离和提纯药物污染内毒素类/分离和提纯大肠杆菌/遗传学大肠杆菌蛋白质类铁蛋白质类/化学铁蛋白质类/遗传学铁蛋白质类/分离和提纯组氨酸/化学膜蛋白质类/化学膜蛋白质类/遗传学膜蛋白质类/分离和提纯癌基因蛋白质类病毒性/化学癌基因蛋白质类病毒性/遗传学癌基因蛋白质类病毒性/分离和提纯聚乙烯二醇类/分离和提纯蛋白质折叠重组融合蛋白质类/化学重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯溶剂

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Population characteristics of Irish Helicobacter pylori isolates:: a tRNA-associated locus

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IRISH JOURNAL OF MEDICAL SCIENCE 2000年第2期169卷 137-140页

作者： Dundon, WG Marshall, DG O'Moráin, CA Smyth, CJ Univ Dublin Trinity Coll Moyne Inst Prevent Med Dept Microbiol Dublin 2 Ireland

Background A strain-variable transfer RNA-associated-locus (trl) was present in 50% of Irish Helicobacter pylori (H. pylori), isolates and did not correlate with the origin of the isolates. Aim To associate a particular genotype or phenotype to trl status in H. pylori by further screening the isolates from the original study for the presence of known genotypic and phenotypic characteristics. Methods Forty two clinical isolates were screened for the presence of the cagA, vacA, iceA1 acid vapD genes by Southern or DNA dot blot analysis. Western blot analysis was performed using antibodies to CagA, VacA, Lewis X (Le(x)) and Lewis Y (Le(y)). Plasmids were identified by the alkaline lysis method. Results The cagA gene was present in 29 (69%) of isolates screened and 21 (50%) produced the CagA protein. The vacA gene was detected in all of the isolates while VacA was expressed in 71.4%. The iceA1 and vapD loci were detected in 73.8% and 71.4% respectively. Le(x) was expressed in 42.9% and Le(y) in 38.1% of the isolates. Expression of both Lewis antigens was detected in 7.1% while in 30.9% neither antigen was detected. Plasmids were present in 47.6%. There was no association between the trl status of isolates and any of the above. There were no significant associations between the phenotypic and genotypic characteristics studied and peptic ulcer disease or non-ulcer dyspepsia. Conclusion The strain-variable tRNA-associated locus is independent of the vacA/VacA, cagA/CagA, Lewis X, Lewis Y, iceA1, vapD and plasmid status in the population of Irish H. pylori isolates studied.

关键词：细菌蛋白质类/遗传学细菌蛋白质类/分离和提纯基因型幽门螺杆菌/遗传学幽门螺杆菌/分离和提纯爱尔兰表型 RNA 细菌/遗传学 RNA 转移/遗传学 RNA 转移甘氨酸/遗传学 RNA 转移亮氨酸/遗传学人类

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Cloning of a new Bacillus thuringiensis cry1I-type crystal protein

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CURRENT MICROBIOLOGY 2000年第1期41卷 65-69页

作者： Choi, SK Shin, BS Kong, EM Rho, HM Park, SH KRIBB Genome Res Ctr Taejon 305600 South Korea KRIBB Lab Microbial & Bioproc Engn Taejon 305600 South Korea Seoul Natl Univ Dept Mol Biol Seoul 151742 South Korea Seoul Natl Univ Res Ctr Cell Differentiat Seoul 151742 South Korea

A new cvy1I-type gene, cry1Id1, was cloned from a B. thuringiensis isolate, and its nucleotide sequence was determined. The deduced amino acid sequence of Cry1Id1 is 89.7%, 87.2%, and 83.4% identical to the Cry1Ia, Cry1Ib, and Cry1Ic proteins, respectively. The upstream sequence of the cry1Id1 structural gene was not functional as promoter in B. subtilis. The Cry1Id1 protein, purified from recombinant E. coli cells, had a toxicity comparable to that of Cry1Ia against Plutella xylostella, but it was significantly less active than Cry1Ia against Bombyx mori. Cry1Id1 was not active against the coleopteran insect, Agelastica coerulea.

关键词：氨基酸序列芽孢杆菌苏云金/遗传学芽孢杆菌苏云金/代谢细菌蛋白质类/遗传学细菌蛋白质类/分离和提纯细菌毒素类/遗传学细菌毒素类/分离和提纯碱基序列甲虫蚕属克隆分子内毒素类/遗传学内毒素类/分离和提纯溶血素蛋白质类杀虫药/分离和提纯分子序列数据蛾蛋白质结构三级动物

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Expression, purification, crystallization and preliminary X-ray diffraction studies of bacterial and archaeal L4 ribosomal proteins

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ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 2000年第5期56卷 645-647页

作者： Worbs, M Wahl, MC Max Planck Inst Biochem Abt Strukturforsch D-82152 Martinsried Germany

Ribosomal protein L4 is implicated in the peptidyltransferase activity of the ribosome and in certain bacteria it regulates the transcription and translation of the 11-gene S10 operon. The genes for the L4 ribosomal proteins from the hyperthermophilic bacterium Thermotoga maritima and the halophilic archaeon Haloarcula marismortui have been PCR amplified from genomic DNA and cloned under the control of a T7 promoter to generate overexpressing Escherichia coli strains. For both proteins, efficient purification procedures were developed to yield material suitable for crystallization trials. Crystals of T. maritima L4 were obtained in the orthorhombic space group P2(1)2(1)2(1), with one molecule per asymmetric unit, diffracting to 1.7 Angstrom resolution with synchrotron radiation. Crystals of H. marismortui L4 belonged to the trigonal space group P3(1)21 or P3(2)21 and diffracted to 3.2 Angstrom resolution with a rotating-anode source, presumably containing three molecules per asymmetric unit. The results demonstrate that for certain halophilic proteins the same purification and crystallization procedures can be employed as for conventional proteins.

关键词：古细菌蛋白质类/化学古细菌蛋白质类/遗传学古细菌蛋白质类/分离和提纯细菌蛋白质类/化学细菌蛋白质类/遗传学细菌蛋白质类/分离和提纯克隆分子结晶结晶学 X线/方法死海嗜盐古细菌/遗传学蛋白质构象重组蛋白质类/化学重组蛋白质类/分离和提纯核糖体蛋白质类/化学核糖体蛋白质类/遗传学核糖体蛋白质类/分离和提纯喜温海洋杆菌/遗传学

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IDENTIFICATION OF A SOLUBLE-PROTEIN THAT STIMULATES PEPTIDE-BOND SYNTHESIS

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1975年第11期72卷 4257-4260页

作者： GLICK, BR GANOZA, MC UNIV TORONTO BANTING & BEST DEPT MED RESTORONTO M5G 1L6ONTARIOCANADA

A soluble protein factor was isolated, free of elongation factor (EF)-T and EF-G, based on its ability to stimulate the synthesis of peptide bonds using ribosomal bound 70S-AUG-N-formyl-[35S]methionyl-tRNA complex and added puromycin as substrates. Over 90% of this activity was found in the ribosome-free cytoplasm of Escherichia coli extracts. Otherfeatures such as molecular weight, purification properties, and catalytic activities distinguish this factor from ribosomal proteins and known activators of translation. The factor requires all components needed for peptide bond synthesis and is inhibited by antibiotics known to specifically block the peptidyl transferase activity of ribosomes. The factor increases the binding affinity of the ribosome for the aminoacyl-tRNA analog puromycin about 10-fold. We suggest that this extraribosomal factor modulates the intrinsic activity of ribosomes to catalyze peptide-bond synthesis, and regard it as a new factor required for peptide chain elongation, which we call EF-P.

关键词：细菌蛋白质类/分离和提纯细菌蛋白质类/药理学酶激活大肠杆菌动力学肽链延伸翻译/药物作用肽基转移酶类/拮抗剂和抑制剂肽基转移酶类/代谢嘌呤霉素/代谢 RNA 转移/代谢核糖体/代谢溶解度

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Cloning, expression and immunological reactivity of two mammalian cell entry proteins encoded by the mce1 operon of Mycobacterium tuberculosis

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SCANDINAVIAN JOURNAL OF IMMUNOLOGY 1999年第5期50卷 510-518页

作者： Ahmad, S Akbar, PK Wiker, HG Harboe, M Mustafa, AS Kuwait Univ Fac Med Dept Microbiol Safat 13110 Kuwait Univ Oslo Natl Hosp Inst Immunol Oslo Norway Natl Inst Publ Hlth Dept Environm Med Sect Environm Immunol Oslo Norway

The DNA segments corresponding to two members of the mammalian cell entry operon 1 (mce1) encoding Mce1A and Mce1E proteins were amplified from Mycobacterium tuberculosis genomic DNA by polymerase chain reaction, cloned and subcloned into pGEM-T and pGEX-4T-3 vectors, respectively, and expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST) of Schistosoma japonicum as the fusion partner. The recombinant proteins appeared as major cellular proteins in SDS-PAGE gels at the expected molecular mass of 68 kDa and 64 kDa for GST-Mce1A and GST-Mce1E, respectively. The identity of each fusion protein was confirmed by reactivity with anti-GST antibodies in Western immunoblots. The fusion proteins were purified to near homogeneity by affinity chromatography, and purified Mce1A and Mce1E, free of the fusion partner, were recovered following specific proteolytic cleavage of the GST portion by thrombin protease. Purified Mce1E appeared as a single band of 38 kDa, whereas purified Mce1A tended to exist in degraded as well as aggregated forms of different sizes. The fusion proteins, free GST and monomeric Mce1A and Mce1E reacted in Western immunoblots with antibodies in pools of human sera from six to 11 tuberculosis patients. Similar analysis showed the presence of antibodies to GST and Mce1A, in pools of human sera from M. bovis BCG-vaccinated healthy subjects. When pure Mce1E was blotted against individual sera, antibodies in 4/10 sera from tuberculosis patients reacted, whereas no reaction was seen with 10 individual sera from M. bovis BCG-vaccinated healthy subjects. However, when the same sera were tested for reactivity to the purified preparation of Mce1A, 8/10 sera from both tuberculosis patients and M. bovis BCG-vaccinated healthy subjects showed positive reactivity. These findings demonstrate that both Mce1A and Mce1E are expressed and immunogenic during natural infection with M, tuberculosis.

关键词：卡介苗/免疫学细菌蛋白质类/遗传学细菌蛋白质类/免疫学细菌蛋白质类/分离和提纯碱基序列克隆分子 DNA引物/遗传学大肠杆菌/遗传学基因表达基因细菌免疫化学结核分枝杆菌/遗传学结核分枝杆菌/免疫学结核分枝杆菌/致病力操纵子结核/免疫学人类

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Cloning, purification, crystallization and preliminary crystallographic analysis of Bacillus subtilis LuxS

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ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY 2001年第9期57卷 1324-1325页

作者： Das, SK Sedelnikova, SE Baker, PJ Ruzheinikov, SN Foster, S Hartley, A Horsburgh, MJ Rice, DW Univ Sheffield Krebs Inst Biomolec Res Dept Mol Biol & Biotechnol Sheffield S10 2TN S Yorkshire England

LuxS of Bacillus subtilis is a member of a novel family of proteins with a potential role in quorum sensing, controlling important aspects of cellular physiology in a range of microbial species. B. subtilis luxS was cloned, expressed in Escherichia coli, purified and crystallized using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant. The crystals belong to one of the enantiomorphic space groups P6(1)22 or P6(5)22, with approximate unit-cell parameters a = b = 63.6, c = 151.5 Angstrom and one subunit in the asymmetric unit, corresponding to a packing density of 2.5 Angstrom (3) Da(-1). The crystals diffract X-rays to at least 1.55 Angstrom resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the key determinants of function of this class of proteins, for which no structures are currently available.

关键词：枯草芽孢杆菌/化学细菌蛋白质类/化学细菌蛋白质类/遗传学细菌蛋白质类/分离和提纯碳硫裂合酶类克隆分子结晶结晶学 X线蛋白质构象重组蛋白质类/化学重组蛋白质类/分离和提纯

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