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J. Biochem. 148, 721–32. A recombinant catalytic domain of matriptase induces detachment and apoptosis of small-intestinal epithelial IEC-6 cells cultured on laminin-coated surface. 2010

Mochida, S., Tsuzuki, S., Inouye, K. and Fushiki, T.

Notes: The authors determined that a recombinant catalytic domain of rat matriptase (His6t-S-CD) caused detachment of small-intestinal epithelial cells (IEC-6 cells) from laminin-coated plates. His6t-S-CD was expressed in the yeast P. pastoris and purified by ammonium sulfate precipitation, gel filtration through a PD-10 column, then Ni2+-chelating chromatography using HisLink™ Resin. The authors also treated IEC-6 cells with purified His6t-S-CD to determine if this domain induced apoptosis by monitoring annexin-V staining, DNA fragmentation and caspase-3 activity. For the DNA fragmentation analysis, IEC-6 cells were treated with His6t-S-CD, then harvested, and genomic DNA was purified using the Wizard® SV Gel and PCR Clean-Up System. DNA fragmentation was assessed by agarose gel electrophoresis and ethidium bromide staining. (4100)

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J. Biol. Chem. 285, 21391–8. LPA19, a Psb27 homolog in Arabidopsis thaliana, facilitates D1 protein precursor processing during PSII biogenesis. 2010

Wei, L., Guo, J., Ouyang, M., Sun, X., Ma, J., Chi, W., Lu, C. and Zhang, L.

Notes: To help understand the molecular mechanism behind the biogenesis and assembly of photosystem II (PSII), the authors characterized the high chlorophyll fluorescence low psii accumulation19 (lpa19) mutation, which results in defective PSII biogenesis. To examine Lpa19 expression patterns in lpa19 mutant plants, the authors performed Western blot analysis using an anti-Lpa19 antibody raised against purified recombinant His-tagged LPA19 protein. This antigen was purified using the HisLink™ Resin. (4101)

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Infect. Immun. 77, 3234–43. Sab, a novel autotransporter of locus of enterocyte effacement-negative shiga-toxigenic Escherichia coli O113:H21, contributes to adherence and biofilm formation. 2009

Herold, S., Paton, J.C. and Paton, A.W.

Notes: To try to understand the mechanism by which certain strains of Shiga-toxigenic E.coli adhere to host intestinal epithelium, the authors characterized the novel autotransporter protein Sab. Expression levels and protein localization were examined by Western blot analysis and enzyme-linked immunosorbent assay. The mouse anti-Sab antibody used in these studies was raised against an N-terminal His6-Sab fusion protein purified using the HisLink™ Resin. (4102)

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Biochem. Biophys. Res. Commun. 372, 929–34. Alternative splicing affecting the SH3A domain controls the binding properties of intersectin 1 in neurons. 2008

Tsyba, L., Gryaznova, T., Dergai, O., Dergai, M., Skrypkina, I., Kropyvko, S., Boldyryev, O., Nikolaienko, O., Novokhatska, O. and Rynditch, A.

Notes: The authors examined protein interactions between splice variants of the SH3A domain of intersection 1 (ITSN1) and the main ITSN1 protein partners using protein pull-down assays. In one set of pull-down assays, SH3A splice variants were expressed as polyhistidine-tagged proteins, and the proline-rich domain of dynamin 1, a known ITSN1 protein partner, was expressed as a glutathione-S-transferase (GST) fusion protein. In a second set of experiments, the SH3A domain variants were expressed as GST-fusion proteins, immobilized, then used to capture endogenous dynamin 1, SOS1, c-Cbl and Cbl-b from cell lysates. Recombinant GST-fusion proteins were purified using glutathione-Sepharose® 4B or the HisLink™ Protein Purification Resin. Based on the data, the authors concluded that alternative splicing of ITSN1 can change the binding properties and its protein partners. (3950)

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J. Biol. Chem. 283, 2478–2487. Roles of functional and structural domains of hepatocyte growth factor activator inhibitor type 1 in the inhibition of matriptase. 2008

Kojima, K., Tsuzuki, S., Fushiki, T. and Inouye, K.

Notes: The authors determined the role of various domains of the hepatocyte growth factor activator inhibitor type 1 (HAI-1) in the inhibition of the protease matriptase. HAI-1 mutants lacking one or more domains were expressed as His-tagged fusion proteins in CHO-K1 or COS-1 cells, and proteins were purified using the HisLink™ Protein Purification Resin. Purified proteins were then used in protease assays with recombinant matriptase. (3788)

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J. Biol. Chem. 282, 7877–7884. Endotoxin-binding proteins modulate the susceptibility of bacterial endotoxin to deacylation by acyloxyacyl hydrolase. 2007

Gioannini, T.L., Teghanemt, A., Zhang, D., Prohinar, P., Levis, E.N., Munford, R.S. and Weiss, J.P.

Notes: The authors examined the deacylation activity of acyloxyacyl hydrolase (AOAH) against a protein:lipooligosaccharides (LOS) complex. A soluble, truncated form of endotoxin-binding protein CD14 (sCD14) was expressed with a hexapolyhistidine tag from a baculovirus vector in High Five insect cells and used to form [3H]LOS:protein complexes. These complexes were incubated with AOAH for 4 hours, then purified using the HisLink™ Protein Purification Resin. The degree of deacylation of the LOS was determined using liquid scintillation spectroscopy. No imidazole was used in the binding buffer or rinses. The buffer did contain 0.1% bovine serum albumin. (3698)

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Mol. Biochem. Parasitol. 156, 235–45. Molecular and biochemical characterization of novel glucokinases from Trypanosoma cruzi and Leishmania spp. 2007

Cáceres, A.J., Quiñones, W., Gualdrón, M., Cordeiro, A., Avilán, L., Michels, P.A. and Concepción, J.L.

Notes: The authors cloned and characterized glucokinases (GlcKs) from Trypanosoma cruzi and Leishmania major. Recombinant GlcKs were expressed in E. coli as His6 fusion proteins and purified using the HisLink™ Protein Purification Resin. Pellets of GlcK-expressing cells were lysed using lysis buffer (100mM Hepes [pH 6.7], 2.0mM MgCl2, and 10mM imidazole) and a French press, and His6-tagged protein was purified from the cleared lysate as directed by the manufacturer using an elution buffer (100mM Hepes [pH 7.6], 2.0mM MgCl2 and 350mM imidazole. (3916)

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J. Mol. Biol. 372, 1215–26. The crystal structure of Trypanosoma cruzi glucokinase reveals features determining oligomerization and anomer specificity of hexose-phosphorylating enzymes. 2007

Cordeiro, A.T., Cáceres, A.J., Vertommen, D., Concepción, J.L., Michels, P.A. and Versées, W.

Notes: The authors expressed Trypanosoma cruzi glucokinase in E. coli BL21(DE3) cells as a polyhistidine-tagged protein and purified the protein using the HisLink™ Protein Purification Resin. BL21(DE3) cell lysates were generated using a French press, and soluble protein was purified using 1ml of HisLink™ resin as directed by the manufacturer. (3915)

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J. Cell Biol. 179, 75–86. Two regulatory steps of ER-stress sensor Ire1 involving its cluster formation and interaction with unfolded proteins. 2007

Kimata, Y., Ishiwata-Kimata, Y., Ito, T., Hirata, A., Suzuki, T., Oikawa, D., Takeuchi, M. and Kohno, K.

Notes: The authors examined the mechanism by which the signal transducer Ire1 senses the accumulation of unfolded proteins during endoplasmic reticulum stress. The authors tested the hypothesis that the core stress-sensing region (CSSR) of Ire1 binds to unfolded proteins by monitoring the ability of purified CSSR and mutant CSSRs to prevent aggregation of denatured firefly luciferase and porcine citrate synthase. CSSR and CSSR mutants were expressed as maltose-binding protein fusions or His8-tagged proteins; the His8-CSSR was expressed in BL21(DE3) cells and purified using HisLink™ Protein Purification Resin. Cell pellets from 400ml cultures were resuspended in 15ml of E. coli lysis buffer (50mM Hepes, pH 8.0, 300mM KCl,
5mM MgCl2, 10mM imidazole, 1% Triton X-100, 2mM phenylmethylsulfonyl fluoride, 0.4mg/ml benzamidine, 0.4mg/ml pepstatin A, 0.4mg/ml leupeptin, 0.3mg/ml lysozyme and 14U/ml DNase I), then disrupted by ultrasonication. Lysates were clarified by centrifugation and incubated with 0.5ml of HisLink™ Resin for 12 hours. The resin was packed into an 8mm-diameter column and sequentially washed with 1) 50mM Hepes (pH 8.0), 1M KCl, 5mM MgCl2, and 0.1% Triton-X 100; 2) 50mM Hepes, 300mM KCl, 5mM MgCl2, 0.1% Triton X-100, and 20mM imidazole; 3) 50mM Hepes, 300mM KCl, 5mM MgCl2, 0.1% Triton X-100, and 40mM imidazole; 4) 50mM Hepes, 300mM KCl, 5mM MgCl2, 0.1% Triton X-100, and 60mM imidazole; 5) 50mM Hepes, 300mM KCl, 5mM MgCl2, and 10mM ATP; and finally 20mM Hepes, 100mM KCl, 5mM MgCl2, and 50% (vol/vol) glycerol. Bound proteins were eluted with 50mM Hepes, 100mM KCl, 5mM MgCl2, 200mM imidazole and 50% (vol/vol) glycerol. (3914)

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Appl. Environ. Microbiol. 72, 986–993. Identification of an endo-ß-1,4-D-xylanase from Magnaporthe grisea by gene knockout analysis, purification, and heterologous expression. 2006

Wu, S.C., Halley, J.E., Luttig, C., Fernekes, L.M., Gutiérrez-Sanchez, G., Darvill, A.G. and Albersheim, P.

Notes: The authors characterized a cell-wall degrading enzyme, endo-ß-1,4-D-xylanase (XYL-6), from the rice pathogen Magnaporthe grisea. Recombinant XYL-6 with a His6 tag was expressed in Pichia pastoris. A 1-liter culture of electrotransformed Pichia cells was grown at a density (OD600) of 2U at 30°C for 3 days. After induction, culture medium containing secreted proteins was concentrated to 100ml, and the polyhistidine-tagged protein was purified by nickel-chelate affinity column chromatography using the HisLink™ Protein Purification Resin. (3565)

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Protein Expr. Purif. 46, 33–9. Purification and characterization of the L-Ara4N transferase protein ArnT from Salmonella typhimurium. 2006

Bretscher, L.E., Morrell, M.T., Funk, A.L. and Klug, C.S.

Notes: The inner membrane protein 4-amino-4-deoxy-L-arabinose transferase (ArnT) catalyzes the final step in the polymyxin-resistance pathway in S. typhimurium and Escherichia coli. The authors expressed and purified ArnT as a His6-tagged protein in E. coli. Briefly, 2 liters of cells expressing ArnT were grown overnight and induced for 3 hours with 1mM IPTG. A French press and pressure cell were used to lyse cells, and the membrane fraction was pelleted using a high-speed centrifugation. Membrane proteins were extracted using 1% dodecylmaltoside, followed by another high-speed centrifugation. The solubilized membrane fraction was passed through a 3ml Q-Sepharose® column. The flowthrough was incubated with 1ml of HisLink™ Protein Purification Resin overnight with gentle mixing. The HisLink™ Resin was washed with a wash buffer containing 10mM imidazole, and the ArnT protein was eluted in a two-step elution using an elution buffer with 100mM and 200mM imidazole. The Q-Sepharose® column was added to the protein purification scheme to eliminate the copurification of E. coli elongation factor Ef-Tu, which contains a cluster of histidine residues and can bind to the HisLink™ Resin if not previously removed. (3854)

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Acta Cryst. F61, 116–117. Expression, purification and preliminary crystallographic analysis of sucrose phosphate synthase (SPS) from Halothermothrix orenii 2005

Huynh, F., Tan, T.-C., Swaminathan, K. and Patel, B. K. C.

Notes: To learn more about the molecular mechanism of thermostability of sucrose phosphate synthase (SPS) from Halothermothrix orenii, the gene was cloned in the pTrcHisA expression vector by PCR, transformed into E. coli and induced for 4 hours with 1mM IPTG. The cells were lysed by sonication and freeze/thaw cycles, and the bacterial lysate was cleared by centrifugation. The HisLink™ Protein Purification Resin was added to the cleared lysate and incubated for 1 hour with gentle agitation. The mixture was transferred to a chromatography column and washed with at least 50 column volumes of 20mM Tris-HCl (pH 7.5), 500mM NaCl, 10mM imidazole. Recombinant SPS was eluted in 20mM Tris-HCl (pH 7.5), 100mM NaCl, 500mM imidazole and the imidazole removed by diafiltration. The purified protein was then allowed to crystallize and used for X-ray diffraction to determine structure. (3281)

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J. Immunol. 175, 4669–76. Molecular basis of reduced potency of underacylated endotoxins. 2005

Teghanemt, A., Zhang, D., Levis, E.N., Weiss, J.P. and Gioannini, T.L.

Notes: The role of various Neisseria meningitidis serotype B [NMB lipooligosaccharides (LOS)] in TLR4-dependent cell activation was investigated. A known protein binding partner, myeloid differentiation protein 2 (MD-2), was expressed in a baculovirus system with a 6X-His tag and the insect medium incubated with purified hexa-, penta-, and tetra-acylated endotoxins metabolically labeled with [3H] or [14C] acetate. These complexes (containing 0.2–1ng of radiolabeled LOS) were then incubated for 1 hour at 25°C with 100µl of HisLink™ Protein Purification Resin. After the incubation, the resin was centrifuged, the supernatant removed and the resin washed 3–4 times in 500µl PBS containing 5mM imidazole before elution with 2% SDS. The presence of radiolabeled LOS was evaluated by liquid scintillation spectroscopy and results were expressed as the percentage of LOS captured. (3317)

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