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Crystallization and preliminary crystallographic analysis of the Rho-binding domain of bovine Rho-kinase

ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 2000年第8期56卷 1042-1044页

作者： Ihara, K Shimizu, T Maesaki, R Okada, K Amano, M Kaibuchi, K Hakoshima, T Nara Inst Sci & Technol Div Struct Biol Nara 6300101 Japan Nara Inst Sci & Technol Div Signal Transduct Nara 6300101 Japan

Rho-kinase binds to a small GTPase Rho in a GTP-dependent manner and regulates many cytoskeletal events in the cell. The minimum region of bovine Rho-kinase sufficient for Rho-binding was expressed as a fusion protein with glutathione S-transferase. After removal of the glutathione S-transferase, thin plate crystals were obtained. The selenomethionine-substituted protein was introduced and crystallized, as was the native protein. The crystals of the Rho-binding domain of Rho-kinase belong to the space group C2, with unit-cell parameters a = 148.0 (2), b = 26.1 (1), c = 39.6 (1) Angstrom, beta = 90.3 (1)degrees. The crystals diffract to a resolution beyond 1.5 Angstrom.

关键词：结合部位结晶大肠杆菌/遗传学谷胱甘肽转移酶/化学谷胱甘肽转移酶/遗传学谷胱甘肽转移酶/分离和提纯细胞内信号肽和蛋白质类蛋白质结构三级蛋白质丝氨酸苏氨酸激酶/化学蛋白质丝氨酸苏氨酸激酶/遗传学蛋白质丝氨酸苏氨酸激酶/分离和提纯重组融合蛋白质类/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 ρGTP结合蛋白质类/代谢 rho相关激酶类动物牛

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Rapid purification of protein complexes from mammalian cells

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NUCLEIC ACIDS RESEARCH 2000年第12期28卷 E61页

作者： Medina, Dan Moskowitz, Neal Khan, Subarna Christopher, Scott Germino, Joseph Univ Med & Dent New Jersey Robert Wood Johnson Med Sch Dept Med New Brunswick NJ 08901 USA

The evaluation of the protein binding partner(s) of biologically important proteins is currently an area of intense research, especially since the development of the yeast two-hybrid assay. However, not all protein-protein interactions uncovered by this assay are biologically relevant and another confirmatory assay must be performed. Ideally, this assay should be rapid, versatile and performed under conditions which mimic the 'normal' physiological state as closely as possible. Towards this goal, we have constructed two eukaryotic expression vectors that facilitate the purification of a protein of interest, along with any associated proteins, from mammalian cells. These vectors incorporate the following features: (i) a tetracycline-responsive promoter so that the level of protein production can be regulated;(ii) an N-terminal glutathione S-transferase tag or a triple repeat of the HA1 epitope, to facilitate purification of the protein either by glutathione affinity chromatography or immunoprecipitation, respectively, followed by a multiple cloning site;(iii) the gene for the enhanced green fluorescent protein (for detection of the presence of the fusion protein and subcellular localization);(iv) a puromycin marker for the selection of stable transformants;(v) a truncated EBNA protein and oriP sequence for episomal replication of the vector. These latter two features permit expansion of small cultures of transfected cells under puromycin selection, thereby increasing the amount of tagged protein that can be purified. We show that these vectors can be used to direct the doxycycline-inducible expresssion of tagged proteins and to recover tagged CIP1-p21 protein complexes from HeLa cells. Furthermore, we show that these tagged p21-purified complexes contain both cyclin A and Cdk2, which are known to interact with p21, but not beta-actin.

关键词： CDC2-CDC28激酶类克隆分子细胞周期蛋白A/分离和提纯细胞周期蛋白依赖激酶2 周期素依赖激酶抑制剂p21 细胞周期蛋白质依赖激酶类/分离和提纯细胞周期蛋白类/分离和提纯多西环素遗传载体谷胱甘肽转移酶/遗传学谷胱甘肽转移酶/分离和提纯 HeLa细胞大分子物质哺乳动物蛋白质结合蛋白质丝氨酸苏氨酸激酶/分离和提纯蛋白质类/遗传学蛋白质类/分离和提纯重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯时间因素转染动物人类

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MOZ is fused to p300 in an acute monocytic leukemia with t(8;22)

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GENES CHROMOSOMES & CANCER 2000年第2期28卷 138-144页

作者： Chaffanet, M Gressin, L Preudhomme, C Soenen-Cornu, V Birnbaum, D Pébusque, MJ INSERM U119 Oncol Mol Lab Inst Cancerol & Immunol Marseille F-13009 Marseille France Inst J Paoli I Calmettes Lab Biol Tumeurs F-13009 Marseille France Fdn Jean Dausset Paris France Hop Calmettes Hematol Lab Lille France

We report on the fusion of the monocytic leukemia zinc finger protein (MOZ) gene to the adenoviral EIA-associated protein p300 (p300) gene in acute monocytic leukemia M5 associated with a t(8;22)(p11;q13) translocation. We studied two patients with double-color fluorescence in situ hybridization (FISH) using the yeast artificial chromosome 176C9 and the bacterial artificial chromosome clone H59D10 specific to the MOZ and p300 genes, respectively. Both probes were split. in the patients' chromosome metaphase cells, and the two derivative chromosomes were each labeled with both probes. We showed by Southern blot the rearrangement of the MOZ gene, and cloned the fusion transcripts in one patient carrying the t(8;22) by reverse transcription-polymerase chain reaction using MOZ- and p300-specific primers. Both fusion transcripts were expressed. This result defines a novel reciprocal translocation involving Mo acetyltransferases, MOZ and p300, resulting in an: abnormal transcriptional co-activator that could play a critical role in leukemogenesis. Genes Chromosomes Cancer 28: 138-144, 2000. (C) 2000 Wiley-Liss, Inc.

关键词：乙酰基转移酶类/遗传学乙酰基转移酶类/分离和提纯氨基酸序列染色体人 22对/遗传学染色体人 8对/遗传学 E1A相关p300蛋白质基因重排组蛋白酰基转移酶原位杂交荧光白血病单核细胞急性/酶学白血病单核细胞急性/遗传学白血病粒-单核细胞慢性/酶学白血病粒-单核细胞慢性/遗传学分子序列数据核蛋白质类/遗传学核蛋白质类/分离和提纯 RNA 信使/分离和提纯重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯反式激活因子类/遗传学反式激活因子类/分离和提纯易位遗传/遗传学肿瘤细胞培养的人类

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Error-free and error-prone lesion bypass by human DNA polymerase κ in vitro

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NUCLEIC ACIDS RESEARCH 2000年第21期28卷 4138-4146页

作者： Zhang, YB Yuan, FH Wu, XH Wang, M Rechkoblit, O Taylor, JS Geacintov, NE Wang, ZG Univ Kentucky Grad Ctr Toxicol Lexington KY 40536 USA Washington Univ Dept Chem St Louis MO 63130 USA NYU Dept Chem New York NY 10003 USA

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Pol kappa encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that Human Pol kappa is a novel lesion bypass polymerase in vitro. Purified human Pol kappa efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Pol kappa most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Pol kappa was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Pol kappa effectively bypassed a template (-)-trans-antibenzo[a]pyrene-N-2-dG lesion in an error-free manner by-incorporating a C opposite the bulky adduct. In contrast, human Pol kappa was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Poi kappa plays an important role in both error-free and error-prone lesion bypass in humans.

关键词： 2-乙酰氨基芴/药理学碱基错配/遗传学碱基序列苯并芘/药理学催化作用 DNA加合物/药物作用 DNA加合物/遗传学 DNA加合物/代谢 DNA损伤/药物作用 DNA损伤/遗传学指导DNA的DNA聚合酶鸟嘌呤/类似物和衍生物鸟嘌呤/代谢诱变/药物作用诱变/遗传学诱变剂/药理学核苷酸类/遗传学核苷酸类/代谢蛋白质类/遗传学蛋白质类/分离和提纯蛋白质类/代谢嘧啶二聚物/遗传学嘧啶二聚物/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/代谢底物特异性模板遗传人类

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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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Nuclear localization signals of the BRCA2 protein

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2000年第1期270卷 171-175页

作者： Yano, K Morotomi, K Saito, H Kato, M Matsuo, F Miki, Y Japanese Fdn Canc Res Inst Canc Dept Mol Diag Toshima Ku Tokyo 1708455 Japan

BRCA2 is a tumor suppressor gene whose germline mutations increase the lifetime risk of breast cancer. BRCA2 encodes a large nuclear protein involved in DNA repair, but the location of its functional domain has been unclear. Here, we report nuclear localization signals (NLSs) of the BRCA2 protein. By expressing various portions of the BRCA2 protein tagged with enhanced green fluorescent protein in HeLa cells, we show that the C-terminal domain is necessary for nuclear localization. Two regions in the C-terminal domain were identified with functional NLSs by site-directed mutagenesis analyses. The NLSs locate between the germline mutation found in the most downstream position and the polymorphic stop codon, suggesting that defects in the proper nuclear transport of the BRCA2 protein are causative of carcinogenesis. Our data thus provide a possible explanation for the high frequency of frame-shift and nonsense mutations in BRCA2 of hereditary breast cancer patients. (C) 2000 Academic Press.

关键词： BRCA2蛋白质生物转运乳腺肿瘤/遗传学细胞区室化基因肿瘤抑制绿色荧光蛋白质类 HeLa细胞发光蛋白质类肿瘤蛋白质类/遗传学肿瘤蛋白质类/分离和提纯核定位信号核蛋白质类/遗传学核蛋白质类/分离和提纯肽碎片/遗传学肽碎片/分离和提纯重组融合蛋白质类/分离和提纯转录因子/遗传学转录因子/分离和提纯女(雌)性人类

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Synthesis of neoglycoenzymes with homogeneous N-linked oligosaccharides using immobilized endo-β-N-acetylglucosaminidase A

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2000年第1期267卷 134-138页

作者： Fujita, K Tanaka, N Sano, M Kato, I Asada, Y Takegawa, K Kagawa Univ Fac Agr Dept Life Sci Kagawa 7610795 Japan Takara Shuzo Co Ltd Biotechnol Res Lab Otsu Shiga 5202193 Japan

A procedure for the enzymatic synthesis of neoglycoenzymes is described. The gene encoding endo-beta-N-acetylglucosaminidase from Arthrobacter proto-phormiae (Endo-A) was overexpressed in Escherichia coli as a fusion protein linked to glutathione S-transferase (GST). GST-Endo-A fusion was extracted as a soluble protein. The fusion protein was purified to homogeneity with glutathione-Sepharose 4B and showed transglycosylation activity toward high-mannose-type glycopeptides without removing the GST moiety. The GST-Endo-A immobilized on glutathione-Sepharose 4B retained its transglycosylation activity. The immobilized enzyme could transfer (Man)(6)GlcNAc en bloc to partially deglycosylated ribonuclease B without damaging its enzyme activity. The immobilized GST-Endo-A should be very useful for synthesizing active neoglycoenzymes attached with homogeneous N-linked oligosaccharides. (C) 2000 Academic Press.

关键词：分子序列数据寡糖类/化学胰腺/酶学重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢核糖核酸酶类/分离和提纯核糖核酸酶类/代谢

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Distinct pathways of cell migration and antiapoptotic response to epithelial injury: Structure-function analysis of human intestinal trefoil factor

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MOLECULAR AND CELLULAR BIOLOGY 2000年第13期20卷 4680-4690页

作者： Kinoshita, K Taupin, DR Itoh, H Podolsky, DK Massachusetts Gen Hosp Gastrointestinal Unit Boston MA 02114 USA Massachusetts Gen Hosp Ctr Study Inflammatory Bowel Dis Boston MA 02114 USA Harvard Univ Sch Med Boston MA 02114 USA

The trefoil peptide intestinal trefoil factor (ITF) plays a critical role in the protection of colonic mucosa and is essential to restitution after epithelial damage. These functional properties are accomplished through coordinated promotion of cell migration and inhibition of apoptosis. ITF contains a unique three-looped trefoil motif formed by intrachain disulfide bonds among six conserved cysteine residues, which is thought to contribute to its marked protease resistance. ITF also has a seventh cysteine residue, which permits homodimer formation. A series of cysteine-to-serine substitutions and a C-terminally truncated ITF were made by PCR site-directed mutagenesis. Any alteration of the trefoil motif or truncation resulted in loss of protease resistance. However, neither an intact trefoil domain nor dimerization was required to promote cell migration. This pro-restitution activity correlated with the ability of the ITF mutants to activate mitogen-activated protein (MAP) kinase independent of phosphorylation of the epidermal growth factor (EGF) receptor. In contrast, only intact ITF retained both phosphatidylinositol 3-kinase and the EGF receptor-dependent antiapoptotic effect in HCT116 and IEC-6 cells. The inability to block apoptosis correlated with a loss of trefoil peptide-induced transactivation of the EGF receptor or Akt kinase in HT-29 cells. In addition to defining structural requirements for the functional properties of ITF, these findings demonstrate that distinct intracellular signaling pathways mediate the effects of ITF on cell migration and apoptosis.

关键词：氨基酸序列细胞凋亡/药物作用细胞凋亡/生理学细胞运动/生理学结肠肿瘤酶抑制剂/药理学上皮/损伤上皮/病理学类黄酮物质/药理学生长物质/化学生长物质/药理学生长物质/生理学丝裂原活化蛋白激酶1/拮抗剂和抑制剂丝裂原活化蛋白激酶1/代谢丝裂原活化蛋白激酶3 丝裂原激活蛋白激酶类/拮抗剂和抑制剂丝裂原激活蛋白激酶类/代谢分子序列数据黏蛋白类肌蛋白质类神经肽类胃蛋白酶A/代谢肽类/化学肽类/药理学肽类/生理学磷酰化蛋白质丝氨酸苏氨酸激酶原癌基因蛋白质类/药物作用原癌基因蛋白质类/代谢原癌基因蛋白质c-akt 重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯信号传导构效关系硫氧还蛋白质类/遗传学硫氧还蛋白质类/分离和提纯胰蛋白酶/代谢肿瘤细胞培养的动物人类大鼠

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Overexpression and purification of human XPA using a Baculovirus expression system

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PROTEIN EXPRESSION AND PURIFICATION 2000年第1期19卷 1-11页

作者： Hermanson, IL Turchi, JJ Wright State Univ Dept Biochem & Mol Biol Sch Med Dayton OH 45435 USA

The xeroderma pigmentosum group A protein (XPA) is an essential component of the eukaryotic nucleotide excision repair (NER) process. Recombinant human. XPA was expressed in baculovirus-infected insect cells as a [His](6)-tagged fusion protein. A two-column purification procedure resulted in greater than 90% purity for the recombinant protein with a final yield of 0.53 mg from 200 ml of infected cells, The recombinant protein migrated as a doublet of 44 and 42 kDa upon SDS-PAGE consistent with that observed for the native protein, XPA can interact with a number of proteins including replication protein A (RPA) which has been implicated in the initial recognition of damaged DNA. Using a modified ELISA, we demonstrate that the recombinant XPA fusion protein also forms a complex with RPA independent of DNA, The ability of XPA to bind damaged DNA was assessed in an electrophoretic mobility shift assay using globally cisplatin-damaged DNA. The results revealed a slight preference for DNA damaged with cisplatin consistent with its proposed role in the recognition of damaged DNA. The recombinant XPA fusion protein was able to complement cell-free extracts immunodepleted of XPA restoring NER-catalyzed incision of cisplatin-damaged DNA in an in vitro excision repair assay. (C) 2000 Academic Press.

关键词：杆状病毒科/遗传学细胞系顺铂/药理学交联试剂/药理学 DNA/代谢 DNA损伤 DNA修复 DNA结合蛋白质类/遗传学 DNA结合蛋白质类/分离和提纯 DNA结合蛋白质类/代谢电泳聚丙烯酰氨凝胶酶联免疫吸附测定遗传载体 HeLa细胞昆虫/细胞学蛋白质结合重组融合蛋白质类/遗传学重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢复制蛋白A 干皮病着色性着色性干皮病蛋白A组动物人类

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Recombinant expression of the Candida rugosa lip4 lipase in Escherichia coli

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PROTEIN EXPRESSION AND PURIFICATION 2000年第2期20卷 308-313页

作者： Tang, SJ Sun, KH Sun, GH Chang, TY Lee, GC Natl Taiwan Ocean Univ Inst Marine Biotechnol Keelung 20224 Taiwan Natl Yang Ming Univ Fac Med Technol Taipei 11211 Taiwan Tri Serv Gen Hosp Dept Surg Div Urol Taipei 11100 Taiwan Natl Def Med Ctr Taipei 11100 Taiwan Acad Sinica Inst Bot Taipei 11529 Taiwan

It is difficult to express recombinant Candida rugosa lipases (CRLs) in heterologous systems, since C. rugosa utilizes a nonuniversal serine codon CUG for leucine. In this study, recombinant LIP4 in which all 19 CUG codons had been converted to a universal serine codon was overexpressed in Escherichia coli BL21(DE3). The recombinant LIP4 was found mainly in the inclusion bodies and showed a low catalytic activity. To increase the amount of soluble form and activity of recombinant LIP4, the DNA was fused to the gene for thioredoxin (TrxFus-LIP4) and then expressed in E. coli strain AD494(DE3). This strategy promotes the formation of disulfide bonds in the cytosol and yields enzymatically active forms of LIP4. The purified recombinant TrxFus-LIP4 and LIP4 expressed in AD494(DE3) had the same catalytic profiles. In addition, recombinant LIP4 had higher esterase activities toward long-chain ester and lower lipase activities toward tributyrin, triolein, and olive oil. This system for the expression of fungal lipase in E. coli strain AD494(DE3) is reliable and may produce enzymatically active forms of recombinant lipase without an in vitro refolding procedure. (C) 2000 Academic Press.

关键词：念珠菌属/酶学密码子电泳聚丙烯酰氨凝胶大肠杆菌真菌蛋白质类/遗传学真菌蛋白质类/分离和提纯真菌蛋白质类/代谢脂肪酶/遗传学脂肪酶/分离和提纯脂肪酶/代谢诱变定点重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢丝氨酸/遗传学溶解度底物特异性甘油三酯类/代谢

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