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Use of hydrophobic interaction chromatography to separate recombinant antibody fragments from associated bacterial chaperone protein GroEL

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ANALYTICAL BIOCHEMISTRY 2000年第2期278卷 239-241页

作者： O'Connor, KC Ghatak, S Stollar, BD Tufts Univ Sch Med Sackler Sch Grad Biomed Sci Dept Biochem Boston MA 02111 USA Tufts Univ Sch Med Div Hematol Oncol Dept Med Boston MA 02111 USA

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NifS-directed assembly of a transient [2Fe-2S] cluster within the NifU protein

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2000年第2期97卷 599-604页

作者： Yuvaniyama, P Agar, JN Cash, VL Johnson, MK Dean, DR Univ Georgia Dept Chem Athens GA 30602 USA Univ Georgia Ctr Metalloenzyme Studies Athens GA 30602 USA Virginia Polytech Inst & State Univ Dept Biochem Blacksburg VA 24061 USA

The Nifs and NifU proteins from Azotobacter vinelandii are required for the full activation of nitrogenase. Nifs is a homodimeric cysteine desulfurase that supplies the inorganic sulfide necessary for formation of the Fe-S clusters contained with in the nitrogenase component proteins, NifU has been suggested to complement NifS either by mobilizing the Fe necessary for nitrogenase Fe-S cluster formation or by providing an intermediate Fe-S cluster assembly site. As isolated, the homodimeric NifU protein contains one [2Fe-2S](2+,+) cluster per subunit, which is referred to as the permanent cluster. In this report, we show that NifU is able to interact with Nifs and that a second, transient [2Fe-2S] cluster can be assembled within NifU in vitro when incubated in the presence of ferric ion, L-cysteine, and catalytic amounts of Nifs. Approximately one transient [2Fe-2S] cluster is assembled per homodimer, The transient [2Fe-2S] cluster species is labile and rapidly released on reduction. We propose that transient [2Fe-2S] cluster units are formed on NifU and then released to supply the inorganic iron and sulfur necessary for maturation of the nitrogenase component proteins. The role of the permanent [2Fe-2S] clusters contained within NifU is not yet known, but they could have a redox function involving either the formation or release of transient [2Fe-2S] cluster units assembled on NifU, Because homologs to both NifU and Nifs, respectively designated IscU and IscS, are found in non-nitrogen fixing organisms, it is possible that the function of NifU proposed here could represent a general mechanism for the maturation of Fe-s cluster-containing proteins.

关键词：氨基酸取代维涅兰德固氮菌/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/分离和提纯铁/化学铁硫蛋白质类/化学大分子物质氧化还原分光光度法紫外线光谱分析拉曼

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Biological and molecular characterization of a two-peptide lantibiotic produced by Lactococcus lactis IFPL105

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JOURNAL OF APPLIED MICROBIOLOGY 2000年第2期89卷 249-260页

作者： Martínez-Cuesta, MC Buist, G Kok, J Hauge, HH Nissen-Meyer, J Peláez, C Requena, T CSIC Inst Frio Dept Dairy Sci & Technol E-28040 Madrid Spain Univ Groningen Groningen Biomol Sci & Biotechnol Inst Dept Genet NL-9700 AB Groningen Netherlands Univ Oslo Dept Biochem N-0316 Oslo Norway

The lactic acid bacterium Lactococcus lactis IFPL105 secretes a broad spectrum bacteriocin produced from the 46 kb plasmid pBAC105. The bacteriocin was purified to homogeneity by ionic and hydrophobic exchange and reverse-phase chromatography. Bacteriocin activity required the complementary action of two distinct peptides (alpha and beta) with average molecular masses of 3322 and 2848 Da, respectively. The genes encoding the two peptides were cloned and sequenced and were found to be identical to the ltnAB genes from plasmid pMRC01 of L. lactis DPC3147. LtnA and LtnB contain putative leader peptide sequences similar to the known 'double glycine' type. The predicted amino acid sequence of mature LtnA and LtnB differed from the amino acid content determined for the purified alpha and beta peptides in the residues serine, threonine, cysteine and alanine. Post-translational modification, and the formation of lanthionine or methyllanthionine rings, could partly explain the difference. Hybridization experiments showed that the organization of the gene cluster in pBAC105 responsible for the production of the bacteriocin is similar to that in pMRC01, which involves genes encoding modifying enzymes for lantibiotic biosynthesis and dual-function transporters. In both cases, the gene clusters are flanked by IS946 elements, suggesting an en bloc transposition. The findings from the isolation and molecular characterization of the bacteriocin provide evidence for the lantibiotic nature of the two peptides.

关键词：氨基酸序列氨基酸类/分析细菌蛋白质类/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/分离和提纯细菌素类印迹法 DNA 基因细菌乳乳球菌/遗传学乳乳球菌/代谢分子序列数据多基因族肽类/化学转录遗传

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Role of Bacillus subtilis SpoIIIE in DNA transport across the mother cell-prespore division septum

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SCIENCE 2000年第5493期290卷 995-997页

作者： Bath, J Wu, LJ Errington, J Wang, JC Harvard Univ Dept Mol & Cellular Biol Cambridge MA 02138 USA Univ Oxford Sir William Dunn Sch Pathol Oxford OX1 3RE England

The SpoIIIE protein of Bacillus subtilis is required for chromosome segregation during spore formation. The COOH-terminal cytoplasmic part of SpoIIIE was shown to be a DNA-dependent adenosine triphosphatase (ATPase) capable of tracking along DNA in the presence of ATP, and the NH2-terminal part of the protein was found to mediate its Localization to the division septum. Thus, during sporulation, SpoIIIE appears to act as a DNA pump that actively moves one of the replicated pair of chromosomes into the prespore. The presence of SpoIIIE homologs in a broad range of bacteria suggests that this mechanism for active transport of DNA may be widespread.

关键词：腺苷三磷酸酶类/代谢腺苷三磷酸/代谢氨基酸特征结构枯草芽孢杆菌/代谢细菌蛋白质类/化学细菌蛋白质类/分离和提纯细菌蛋白质类/代谢生物转运主动 DNA解旋酶类 DNA拓扑异构酶类 Ⅰ型/代谢 DNA 细菌/代谢 DNA 超螺旋/代谢重组蛋白质类/化学重组蛋白质类/分离和提纯重组蛋白质类/代谢 ∑因子孢子细菌/代谢转录因子

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Sulfite:Cytochrome c oxidoreductase from Thiobacillus novellus -: Purification, characterization, and molecular biology of a heterodimeric member of the sulfite oxidase family

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第18期275卷 13202-13212页

作者： Kappler, U Bennett, B Rethmeier, J Schwarz, G Deutzmann, R McEwan, AG Dahl, C Univ Bonn Inst Mikrobiol & Biotechnol D-53115 Bonn Germany CCLRC Daresbury Lab Warrington WA4 4AD Cheshire England Univ Bremen UFT D-28334 Bremen Germany Univ Bremen Fachbereich 2 D-28334 Bremen Germany Tech Univ Carolo Wilhelmina Braunschweig Biozentrum D-38106 Braunschweig Germany Univ Regensburg Inst Biochem D-93053 Regensburg Germany Univ Queensland Dept Microbiol & Parasitol St Lucia Qld 4072 Australia

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC 1.8.2.1), Here we show that the enzyme from Thiobacillus novellus is a periplasmically located alpha beta heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa monoheme cytochrome c(552) smbunit (midpoint redox potential, Em(8.0) = +280 mV), The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K-m values for sulfite and cytochrome c(550) were determined to be 27 and 4 mu M, respectively;the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorAB genes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any known c-type cytochromes.

关键词：氨基酸序列细菌蛋白质类/化学细菌蛋白质类/遗传学细菌蛋白质类/分离和提纯细菌蛋白质类/代谢碱基序列细胞色素还原酶/化学细胞色素还原酶/遗传学细胞色素还原酶/分离和提纯细胞色素还原酶/代谢分子序列数据序列比对底物特异性亚硫酸脱氢酶硫杆菌属/酶学

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High resolution electroelution of polyacrylamide gels for the purification of single proteins from Mycobacterium tuberculosis culture filtrate

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SCANDINAVIAN JOURNAL OF IMMUNOLOGY 2000年第1期51卷 79-86页

作者： Weldingh, K Hansen, A Jacobsen, S Andersen, P Statens Serum Inst Dept TB Immunol DK-2300 Copenhagen S Denmark Tech Univ Denmark Dept Biochem & Nutr DK-2800 Lyngby Denmark

Culture filtrate from Mycobacterium tuberculosis contains protective molecules which have been used successfully in experimental vaccines against tuberculosis. Despite an increasing number of mycobacterial proteins being characterised, a major effort is still needed to get an overview of the many potentially interesting molecules in culture filtrate. In this study we describe a high throughput method for purification and biological evaluation of protein components in complex protein mixtures. The method presents a new application of the recently developed Mini Whole Gel fluter and employs this apparatus for the high resolution electroelution of selected molecular mass fractions of protein mixtures previously separated in large polyacrylamide gels. Two novel M. tuberculosis culture filtrate proteins (CspA and TB18.6) were purified by this method, their N-terminal sequences were determined and the open reading frame encoding each of the proteins identified. The immunological recognition of the molecules were evaluated in tuberculosis infected mice and guinea pigs. Both proteins induced DTH responses in guinea pigs and IFN-gamma release from spleen lymphocytes isolated from infected mice.

关键词：氨基酸序列抗原细菌/化学抗原细菌/遗传学抗原细菌/分离和提纯细菌蛋白质类/遗传学细菌蛋白质类/免疫学细菌蛋白质类/分离和提纯电泳聚丙烯酰氨凝胶/方法超敏反应迟发型干扰素γ/代谢淋巴细胞/免疫学小鼠近交C57BL 小鼠近交CBA 分子序列数据分子量结核分枝杆菌/化学结核分枝杆菌/遗传学结核分枝杆菌/免疫学序列同源性氨基酸结核/免疫学动物女(雌)性豚鼠小鼠

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Electrophoresis of proteins and protein-protein complexes in a native agarose gel

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ANALYTICAL BIOCHEMISTRY 2000年第1期282卷 147-149页

作者： Kim, R Yokota, H Kim, SH Univ Calif Berkeley Lawrence Berkeley Lab Div Phys Biocsci Berkeley CA 94720 USA Univ Calif Berkeley Dept Chem Berkeley CA 94720 USA

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Triton X-114 phase partitioning for the isolation of a pediocin-like bacteriocin from Carnobacterium divergens

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LETTERS IN APPLIED MICROBIOLOGY 2000年第1期30卷 42-46页

作者： Métivier, A Boyaval, P Duffes, F Dousset, X Compoint, JP Marion, D INRA Lab Biochim & Technol Prot Nantes 03 France INRA Rech Technol Laitiere Lab F-35042 Rennes France ENITIAA Microbiol Lab Nantes France

A new procedure combining Triton X-114 phase partitioning and cation exchange chromatography was developed to purify a bacteriocin from a complex culture medium. This pediocin-like bacteriocin, secreted by Carnobacterium divergens and named divercin V41, was entirely recovered in the lower detergent-rich phase whereas all other substances (compounds from culture medium, bacterial metabolites) remained in the upper detergent-poor phase. Subsequent cation-exchange chromatography of the TX-114-rich phase allowed recovery of the pure active bacteriocin and also detergent removing. This new purification method is versatile, fast (only two steps) and call be carried out on whole broth.

关键词：细菌/代谢细菌蛋白质类/分离和提纯细菌素类/分离和提纯色谱法高压液相色谱法离子交换培养基/化学洗涤剂聚乙烯二醇类

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Expression of active monomeric and dimeric nuclease A from the gram-positive Streptococcus gordonii surface protein expression system

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PROTEIN EXPRESSION AND PURIFICATION 2000年第1期19卷 158-172页

作者： Dutton, EK Ottum, SA Bolken, TC Franke, CA Hruby, DE SIGA Res Labs Corvallis OR 97333 USA Oregon State Univ Dept Microbiol Corvallis OR 97331 USA

We used the surface protein expression (SPEX) system to express an anchored and a secreted form of staphylococcal nuclease A (NucA) from gram-positive bacteria. NucA is a small (similar to 18 kDa), extracellular, monomeric enzyme from Staphylococcus aureus, A deletion of amino acids 114-119 causes monomeric NucA to form homodimers, The DNA sequence encoding either wild-type or deletion mutant NucA was cloned via homologous recombination into Streptococcus gordonii. S. gordonii strains expressing either anchored or secreted, monomeric or dimeric NucA were isolated and tested for enzymatic activity using a novel fluorescence enzyme assay, We show that active monomeric and dimeric NucA enzyme can be expressed either anchored on the cell surface or secreted into the culture medium, The activity of the dimer NucA was similar to 100-fold less than the monomer, Secreted and anchored, monomeric NucA migrated on SDS-polyacrylamide gels at similar to 18 or similar to 30 kDa, respectively. In addition, similar to S. aureus NucA, the S, gordonii recombinant NucA enzyme was dependent on CaCl2 and was heat stable. In contrast, however, the recombinant NucA activity was maximal at pH 7.0-7.5 whereas S. aureus NucA was maximal at pH 9.0. These results show, for the first time, expression of active enzyme and polymeric protein in secreted and anchored forms using SPEX, This further demonstrates the utility of this gram-positive surface protein expression system as a potential commensal bacterial delivery system for active, therapeutic enzymes, biopharmaceuticals, or vaccines. (C) 2000 Academic Press.

关键词：细菌蛋白质类/生物合成细菌蛋白质类/遗传学细菌蛋白质类/分离和提纯印迹法蛋白质氯化钙/化学色谱法凝胶 DNA/代谢二聚作用酶稳定性荧光采暖氢离子浓度膜蛋白质类/生物合成膜蛋白质类/遗传学膜蛋白质类/分离和提纯微球菌核酸酶/生物合成微球菌核酸酶/遗传学微球菌核酸酶/分离和提纯分子序列数据蛋白质结构四级重组蛋白质类/生物合成重组蛋白质类/遗传学重组蛋白质类/分离和提纯链球菌属/遗传学

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Crystallization and preliminary X-ray diffraction studies of mutants of B1 IgG-binding domain of protein L from Peptostreptococcus magnus

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ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 2000年第Pt 4期56卷 506-508页

作者： Johnsen, K O'Neill, JW Kim, DE Baker, D Zhang, KYJ Fred Hutchinson Canc Res Ctr Div Basic Sci Seattle WA 98109 USA Univ Washington Dept Biochem Seattle WA 98195 USA

The small 62-residue IgG-binding domain B1 of protein L from Peptostreptococcus magnus (Ppl-B1) has proven to be a simple system for the study of the thermodynamics and kinetics of protein folding. X-ray diffraction studies have been initiated in order to determine how the thermostability, folding and unfolding rates of a series of point mutations spanning Ppl-B1 correlate with the high-resolution structures. To this end, a tryptophan-containing variant of Ppl-B1 (herein known as wild type) and two mutants, Lys61Ala and Val49Ala, have been crystallized. Full data sets have been collected for the wild type and the Lys61Ala and Val49Ala mutants to resolutions of 1.7, 2.3 and 1.8 Angstrom, respectively. interestingly all three crystallize using different precipitants and in different space groups. This may be a consequence of the relatively large effects of single-site mutations on surface-charge distribution or structural conformation, which might affect crystal contact sites.

关键词：丙氨酸氨基酸取代细菌蛋白质类/化学细菌蛋白质类/免疫学细菌蛋白质类/分离和提纯结合部位结合部位抗体结晶结晶学 X线免疫球蛋白G 赖氨酸诱变定点消化链球菌属/化学重组蛋白质类/化学重组蛋白质类/免疫学重组蛋白质类/分离和提纯缬氨酸

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