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Genetic and transcriptional analysis of a regulatory region in streptococcal conjugative transposon Tn5252

PLASMID 2000年第3期44卷 262-274页

作者： Srinivas, P Vijayakumar, MN Oklahoma State Univ Dept Microbiol & Mol Genet Stillwater OK 74078 USA

In an attempt to increase our understanding of the mechanisms of conjugal transposition among gram-positive bacteria, we analyzed the genetic and structural properties of 1.2-kb DNA fragment at the left end of the streptococcal conjugative transposon Tn5252. The sequence data revealed four short open reading frames. Polypeptides likely to correspond to two of these genes were identified. Transcriptional start sites and the promoter sequences of three transfer-related genes in the left terminal region of the element were identified. The deduced amino acid sequence of one of these. ORF3, was found to be similar to that of several prokaryotic transcriptional regulator proteins. insertion mutagenesis at this locus reduced the transfer of the element by three orders of magnitude. The presence of a multicopy plasmid carrying ORF3 in a donor cell carrying Tn5252 with a mutated copy of ORF3 or an unaltered element also reduced the transfer frequency of the element similarly. Gel mobility shift assays showed that the ORF3 protein was capable of binding to not only other discrete sites at the left end of the element but also its own promoter, suggesting autoregulation. These results indicate that the ORF3 protein is involved in the regulation of the conjugative transposition of the element. (C) 2000 Academic Press.

关键词：氨基酸序列细菌蛋白质类/分析碱基序列接合遗传 DNA可移植因子/遗传学 DNA 细菌/遗传学电泳琼脂凝胶电泳聚丙烯酰氨凝胶遗传载体分子序列数据突变重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯限制性内切酶图谱法序列比对序列同源性氨基酸链球菌属/遗传学

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Expression and purification of recombinant human annexin V in Escherichia coli

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PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 2000年第4期30卷 305-312页

作者： Zhang, LN Yang, X Hua, ZC Nanjing Univ Dept Biochem State Key Lab Pharmaceut Biotechnol Nanjing 210093 Peoples R China

Human annexin V cDNA was cloned into plasmid pET19b and fused to a ten consecutive histidine tag at N-terminal. When expressed in E. coli BL21(DE3) LysS, the recombinant His10-annexin V accumulated in soluble form in the cytoplasm. By two-step chromatography, i.e., metal chelate affinity chromatography and anion exchange chromatography, recombinant His10-annexin V was purified to homogenity on silver-stained SDS-PAGE gel. Recombinant annexin V, 7.4 mg, was obtained from a 1 litre flask culture.

关键词：膜联蛋白A5/遗传学膜联蛋白A5/分离和提纯膜联蛋白A5/代谢克隆分子 DNA 互补电泳聚丙烯酰氨凝胶酶联免疫吸附测定大肠杆菌/遗传学大肠杆菌/代谢质粒/遗传学质粒/代谢重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢人类

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Facilitated nucleocytoplasmic shuttling of the Ran binding protein RanBP1

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MOLECULAR AND CELLULAR BIOLOGY 2000年第10期20卷 3510-3521页

作者： Plafker, K Macara, IG Univ Virginia Markey Ctr Cell Signaling Charlottesville VA 22908 USA Univ Virginia Dept Pharmacol Charlottesville VA 22908 USA

The Ran binding protein RanBP1 is localized to the cytosol of interphase cells. A leucine-rich nuclear export signal (NES) near the C terminus of RanBP1 is essential to maintain this distribution. We now show that RanBP1 accumulates in nuclei of cells treated with the export inhibitor, leptomycin B! and collapse of the nucleocytoplasmic Ran:GTP gradient lends to equilibration of RanBP1 across the nuclear envelope. Low temperature prevents nuclear accumulation of RanBP1, suggesting that import does not occur via simple diffusion. Glutathione S-transferase (GST)-RanBP1(1-161), which lacks the NES, accumulates in the nucleus after cytoplasmic microinjection. In permeabilized cells, nuclear accumulation of GST-RanBP1 (1-161) requires nuclear Ran:GTP but is not inhibited by a dominant interfering G19V mutant of Ran. Nuclear accumulation is enhanced by addition of exogenous karyopherins/importins or RCC1, both of which also enhance nuclear Ran accumulation. Import correlates with Ran concentration. Remarkably, an E37K: mutant of RanBP1 does not import into the nuclei under any conditions tested despite the fact that it can form a ternary complex with Ran and importin beta. These data indicate that RanBP1 translocates through the pores by an active, nonclassical mechanism and requires Ran:GTP for nuclear accumulation. Shuttling of RanBP1 may function to clear nuclear pores of Ran:GTP, to prevent premature release of import cargo from transport receptors.

关键词：生物转运主动/药物作用载体蛋白质类/代谢细胞区室化细胞周期蛋白质类细胞核/代谢冷温度细胞质/代谢 DNA结合蛋白质类/代谢能量代谢脂肪酸类不饱和/药理学绿色荧光蛋白质类鸟嘌呤核苷酸交换因子类鸟苷三磷酸/代谢核胞浆转运蛋白类发光蛋白质类/遗传学发光蛋白质类/代谢突变核蛋白质类/遗传学核蛋白质类/分离和提纯核蛋白质类/代谢蛋白质结合受体胞质和核重组融合蛋白质类/分离和提纯 ranGTP结合蛋白质/遗传学 ranGTP结合蛋白质/分离和提纯 ranGTP结合蛋白质/代谢

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MDM2 inhibits p300-mediated p53 acetylation and activation by forming a ternary complex with the two proteins

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2000年第23期97卷 12547-12552页

作者： Kobet, E Zeng, XY Zhu, Y Keller, D Lu, H Oregon Hlth Sci Univ Dept Biochem & Mol Biol Portland OR 97201 USA

p300 acetylates and activates the tumor suppressor p53 after DNA damage. Here, we show that MDM2, a negative-feedback regulator of p53, inhibited p300-mediated p53 acetylation by complexing with these two proteins. First, we purified a p300-MDM2-p53 protein complex from HeLa nuclear extracts, which was inactive in p53 acetylation, but active in histone acetylation. Also, wild-type, but not N-terminally deleted, MDM2 inhibited p53 acetylation by p300 in vitro and in vivo. This inhibition was specific for p53, because MDM2 did not affect acetylation of histones or the C terminus of p73 by p300. Consequently, wild-type, but not the mutant, MDM2 repressed the p300-stimulated sequence-specific DNA-binding and transcriptional activities of p53. These results demonstrate that an additional mechanism of p53 inactivation by MDM2 is to inhibit p53 acetylation by p300.

关键词：乙酰化作用乙酰基转移酶类/分离和提纯乙酰基转移酶类/代谢细胞周期蛋白质类/分离和提纯细胞周期蛋白质类/代谢细胞提取物细胞核/代谢 DNA/代谢 γ射线 HeLa细胞组蛋白酰基转移酶核蛋白质类/分离和提纯核蛋白质类/代谢原癌基因蛋白质类/生物合成原癌基因蛋白质类/分离和提纯原癌基因蛋白质类/代谢原癌基因蛋白质c-mdm2 重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢转录因子肿瘤细胞培养的肿瘤抑制蛋白质p53/遗传学肿瘤抑制蛋白质p53/分离和提纯肿瘤抑制蛋白质p53/代谢紫外线动物人类男(雄)性小鼠

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A novel method for expression and large-scale production of human brain L-glutamate decarboxylase

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2000年第3期267卷 777-782页

作者： Davis, KM Foos, T Bates, CS Tucker, E Hsu, CC Chen, WQ Jin, H Tyburski, JB Schloss, JV Tobin, AJ Wu, JY Univ Kansas Dept Mol Biosci Lawrence KS 66045 USA Univ Kansas Dept Med Chem Lawrence KS 66045 USA Univ Calif Los Angeles Dept Biol Los Angeles CA 90024 USA Acad Sinica Inst Biol Chem Taipei Taiwan

L-Glutamate decarboxylase (GAD;EC 4.1.1.15) is the rate-limiting enzyme involved in the synthesis of gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, Imbalance in the conversion of glutamate to GABA has been implicated in a host of human diseases, Studies on the structure, function, and therapeutic use of GAD have been precluded by insufficient quantities of purified active enzyme. Here we report a novel methodology for the expression and large-scale production of enzymatically active, pure, recombinant human GAD65 and GAD67. This method circumvents the sequestering of expressed protein into insoluble inclusion bodies and reduces production of truncated proteins. The availability of sufficient quantities of purified HGAD65 and HGAD67 has allowed for the production of specific polyclonal antibodies that discriminate between the two isoforms. This methodology, in addition to providing key human brain enzymes, may be generally applicable to other systems. (C) 2000 Academic Press.

关键词：脑/酶学克隆分子大肠杆菌谷氨酸脱羧酶/生物合成谷氨酸脱羧酶/遗传学谷氨酸脱羧酶/分离和提纯免疫印迹法同工酶类/生物合成同工酶类/遗传学同工酶类/分离和提纯重组融合蛋白质类/生物合成重组融合蛋白质类/分离和提纯重组蛋白质类/生物合成重组蛋白质类/分离和提纯突触体/酶学人类

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High-yield expression and purification of recombinant proteins in bacteria: A versatile vector for glutathione S-transferase fusion proteins containing two protease cleavage sites

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ANALYTICAL BIOCHEMISTRY 2000年第2期281卷 232-234页

作者： Sehgal, BU Dunn, R Hicke, L Godwin, HA Northwestern Univ Dept Chem Evanston IL 60208 USA Northwestern Univ Dept Biochem Mol Biol & Cell Biol Evanston IL 60208 USA

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Generation of a baculovirus recombinant prostate-specific membrane antigen and its use in the development of a novel protein biochip quantitative immunoassay

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PROTEIN EXPRESSION AND PURIFICATION 2000年第1期19卷 12-21页

作者： Xiao, Z Jiang, X Beckett, ML Wright, GL Eastern Virginia Med Sch Dept Microbiol & Mol Cell Biol Norfolk VA 23507 USA Eastern Virginia Med Sch Ctr Pediat Res Norfolk VA 23507 USA Eastern Virginia Med Sch Virginia Prostate Cr Norfolk VA 23507 USA Sentara Canc Inst Norfolk VA 23507 USA

Prostate-specific membrane antigen (PSMA) is a 100-kDa transmembrane glycoprotein identified by the monoclonal antibody 7E11-C5.3 from the human prostate tumor cell line LNCaP. Because of its significant upregulation in androgen refractory and metastatic prostate cancers, PSMA may be a useful prognostic biomarker and a target for developing novel therapeutic strategies, However, the lack of abundant pure PSMA protein and the low efficacy in immunoaffinity isolation from LNCaP cells have hampered the development of clinical assays. In order to obtain a renewable and reliable source of pure antigen, we used the baculovirus/insect cell system to express and purify a recombinant PSMA. A recombinant baculovirus containing a 6x histidine-tagged PSMA gene was generated, from which rPSMA was expressed and purified using cobalt-chelating affinity chromatography, The purity and correct molecular size of rPSMA were demonstrated by gel electrophoresis and mass spectrometry. Glycosidase digestions showed that the oligosaccharides on rPSMA are primarily N-linked high-mannose type. Although the glycosylation is different from the native PSMA, it did not affect the immunoreactivity of rPSMA to antibodies specific for either the intra- or the extracellular domains of PSMA. Finally, the purified rPSMA was successfully used to develop a quantitative PSMA immunoassay using the novel ProteinChip surface-enhanced laser desorption/ionization mass spectrometry technology. (C) 2000 Academic Press.

关键词：抗体单克隆抗原肿瘤/血液抗原肿瘤/分离和提纯抗原肿瘤/代谢抗原表面杆状病毒科/遗传学印迹法蛋白质羧肽酶类/血液羧肽酶类/分离和提纯羧肽酶类/代谢细胞系色谱法亲和遗传载体谷氨酸羧肽酶Ⅱ 糖基化免疫测定/方法鳞翅目/细胞学重组融合蛋白质类/血液重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢光谱法质量基质辅助激光解吸电离/方法动物人类

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Chimeras of Dictyostelium myosin II head and neck domains with Acanthamoeba or chicken smooth muscle myosin II tail domain have greatly increased and unregulated actin-dependent MgATPase activity

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2000年第23期97卷 12553-12558页

作者： Liu, X Shu, S Yamashita, RA Xu, YH Korn, ED NHLBI Cell Biol Lab NIH Bethesda MD 20892 USA NHLBI Dept Mol Cardiol NIH Bethesda MD 20892 USA

Phosphorylation of the regulatory light chain of Dictyostelium myosin II increases V-max of its actin-dependent MgATPase activity about 5-fold under normal assay conditions. Under these assay conditions, unphosphorylated chimeric myosins in which the tail domain of the Dictyostelium myosin II heavy chain is replaced by either the tail domain of chicken gizzard smooth muscle or Acanthamoeba myosin II are 20 times more active because of a 10- to 15-fold increase in V-max and a 2- to 7-fold decrease in apparent K-ATPase and are only slightly activated by regulatory light chain phosphorylation. Actin-dependent MgATPase activity of the Dictyostelium/Acanthamoeba chimera is not affected by phosphorylation of serine residues in the tail whose phosphorylation completely inactivates wild-type Acanthamoeba myosin II. These results indicate that the actin-dependent MgATPase activity of these myosins involves specific, tightly coupled, interactions between head and tail domains.

关键词：棘阿米巴属/代谢肌动蛋白类/代谢 Ca(2+)Mg(2+)ATP酶/代谢阳离子二价鸡网柱菌属/遗传学网柱菌属/代谢酶激活基因表达镁肌平滑/代谢肌球蛋白重链/化学肌球蛋白重链/遗传学肌球蛋白重链/分离和提纯肌球蛋白重链/代谢肌球蛋白类/遗传学肌球蛋白类/代谢磷酰化蛋白质构象蛋白质结构三级重组融合蛋白质类/化学重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢动物

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Membrane-bound human 3β-hydroxysteroid dehydrogenase:: Overexpression with His-tag using a Baculovirus system and single-step purification

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PROTEIN EXPRESSION AND PURIFICATION 2000年第2期18卷 169-174页

作者： Huang, YW Lu, ML Qi, H Lin, SX CHU Laval Res Ctr MRC Grp Mol Endocrinol Quebec City PQ G1V 4G2 Canada Univ Laval Quebec City PQ G1V 4G2 Canada

The membrane-bound human 3 beta-hydroxysteroid dehydrogenase type 1 (3 beta-HSD1) was overexpressed with His(6)-tag, using a baculovirus expression system, and then purified by nickel-chelated affinity chromatography. Overexpression of 3 beta-HSD1 was confirmed by enzyme assay and Western blot analysis. The protein was purified to more than 95% homogeneity by a single-step Ni2+-chelated affinity chromatography after solubilization of the membrane-bound protein with the detergent C12E8. High yield was repeatedly obtained, with 3-4 mg of homogeneous and active 3 beta-HSD1 from 1 x 10(9) of infected Sf9 cells. The kinetic study showed a K-m of 1.7 mu M and a V-max of 50 nmol/min/mg of purified protein using dehydroepiandrosterone as the substrate. The above preparation will facilitate the structure-function study of this important enzyme. (C) 2000 Academic Press.

关键词：杆状病毒科/遗传学印迹法蛋白质细胞系色谱法亲和电泳聚丙烯酰氨凝胶遗传载体同工酶类/遗传学同工酶类/分离和提纯同工酶类/代谢动力学膜蛋白质类/遗传学膜蛋白质类/分离和提纯膜蛋白质类/代谢重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢斜纹夜蛾属/细胞学斜纹夜蛾属/病毒学动物人类

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Crystallization and preliminary X-ray analysis of insect antifreeze protein from the beetle Tenebrio molitor

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ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 2000年第3期56卷 354-356页

作者： Liou, YC Davies, PL Jia, ZC Queens Univ Dept Biochem Kingston ON K7L 3N6 Canada

Hyperactive antifreeze protein from the beetle Tenebrio molitor (TmAFP) was produced in Escherichia coli and purified by gel-permeation chromatography and HPLC. An iodinated derivative was prepared by incubating the 8.5 kDa TmAFP with N-iodosuccinimide. Native and iodinated TmAFP produced two different crystal Terms when crystallized using the hanging-drop vapor-diffusion technique. Native crystals were rectangular plates that diffracted to similar to 2.5 Angstrom resolution. They were monoclinic and belonged to the space group P2(1), with unit-cell dimensions a = 38.4, b = 73.4, c = 59.3 Angstrom, beta = 97.0 degrees. Crystals of iodinated TmAFP formed elongated hexagons that allowed data to be. collected to similar to 1.4 Angstrom. These crystals belonged Co the space group P6(1) (or P6(5)), with unit-cell dimensions a = 73.85, b = 73.85, c = 53.15 Angstrom. There were two molecules pet asymmetric unit, which corresponds to V-m = 2.46 Angstrom Da(-1) and 51% solvent content. A twofold non-crystallographic symmetry was evident from self-rotation calculations.

关键词：抗冻蛋白质类结晶结晶学 X线大肠杆菌糖蛋白类/化学糖蛋白类/分离和提纯昆虫蛋白质类/化学昆虫蛋白质类/分离和提纯模型分子蛋白质构象重组融合蛋白质类/化学重组融合蛋白质类/分离和提纯拟步行虫属/化学动物

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