Notes: The authors expressed the seven subunits comprising the middle module of Mediator, the 1.4MDa coactivator complex required for regulated transcription by RNA polymerase II, in E. coli. To detect and characterize the interaction of these subunits within the module, they coexpressed combinations of these subunits and performed pull-down assays. This enabled them to publish a subunit interaction map. Coexpression and pull-down assays were performed using proteins purified using MagneHis™ Ni-Particles. (4122)

Notes: The authors identified proteins that bind to small ubiquitin-like modification (SUMO) proteins using a yeast two-hybrid screen. The proteins ability to bind SUMO was confirmed using protein:protein interaction studies. In these studies, recombinant SUMO-1 was expressed with a His₆ tag and immobilized using MagneHis™ Ni-Particles. Putative SUMO-binding proteins were expressed with a GST tag and the proteins were co-incubated to allow them to interact. Proteins bound to immobilized SUMO-1 were eluted with Laemmli sample buffer and detected by Western blot. (4121)

Notes: The protein phosphatase Cdc14 is sequestered in the nucleolus during interphase and, after successful interphase, is dispersed from the nucleolus throughout the cell by an unknown mechanism to drive a cell's exit from mitosis. The authors determined that Cdc14 contains a nuclear localization signal (NLS), but phosphorylation of serine and threonine residues adjacent to the NLS interferes with localization. These phosphorylation sites were mapped using trypsin and AspN digestion, followed by mass spectrometry. The authors showed that phosphorylation of Cdc14 is mediated by the protein kinase Dbf2-Mob1 by co-incubating purified Dbf2-Mob1 and Cdc14 in the presence of γ-[³²P]ATP. Dbf2-Mob1 was expressed with a His6 tag and purified using MagneHis™ Ni-Particles. (4120)

Notes: The authors identify two partial transcripts that encode granulin-like molecules in Aedes albopictus and Manduca sexta. To test the hypothesis that granulin is a highly conserved growth factor that acts on insect cells, the authors expressed recombinant goldfish granulin with an N-terminal His₆ tag, purified the recombinant protein, exposed A. albopictus Aa23 embryonic cells and M. sexta haemocytes to the purified protein, then monitored cell proliferation using a BrdU proliferation assay. Recombinant goldfish granulin was expressed in E. coli and purified using MagneHis™ Ni Particles. (3964)

Notes: The authors developed an in vitro selection system that is based on ribosome display and favors identification of 5´-untranslated regions (UTRs) with high translation efficiencies. A 5´-UTR random library was created in which the 5´-UTRs were upstream of a polyhistidine-tag/Renilla luciferase-coding region. In vitro transcripts from this library were translated in vitro using the Flexi^® Rabbit Reticulocyte Lysate System. The authors preferentially selected mRNAs with high translational efficiencies by shortening the translation time and capturing ternary complexes of mRNA, ribosome and nascent proteins. These complexes were captured using MagneHis™ Ni Particles. RNA was extracted from these complexes and used as a template in RT-PCR for the next round of selection. Before and after each round of selection, 9µl of RNA was translated in vitro, and 20µl of translated product was removed every 5 minutes to measure Renilla luciferase activity and monitor translation efficiency. Renilla luciferase was measured using the Renilla Luciferase Assay System. After two rounds of selection, RT-PCR products were cloned into a pUC18 vector, the sequences of the resulting plasmids were confirmed, and 0.5µg of plasmid was translated in vitro using the TNT^® T7 Coupled Rabbit Reticulocyte Lysate System to further evaluate translation efficiency. (3963)

Notes: The authors used biochemical and mutational analysis to characterize human lysosomal DNaseIIα. Native DNaseIIα and site-directed mutants were expressed as Flag-His₆-DNaseIIα and HA-tagged DNaseIIα using expression constructs created with the pCI Vector. Protein was expressed by transiently transfecting HEK 293-T cells using the TransFast™ Transfection Reagent. Flag-His₆-DNaseIIα was purified using the MagneHis™ Ni-Particles, and this purified protein was used in nuclease assays to monitor catalytic activity and in gel filtration experiments and coimmunoprecipitation assays with HA-DNaseIIα to determine whether the active enzyme is monomeric. (3786)

Notes: This study sought to determine the roles of the tsc1⁺, tsc2⁺ and rhb1⁺ gene products in the starvation response in Schizosaccharomyces pombe. Recombinant Rhb1 was expressed as a polyhistidine-tagged protein in E. coli using the pET30a vector and used as the antigen for polyclonal antibody production. Recombinant Rhb1 was purified using the MagneHis™ Protein Purification System. (3568)

Notes: These authors expressed a recombinant form of a novel hemapoietic granulin from goldfish. This recombinant granulin was expressed with a His₆ tag in a 1-liter culture of BL21 Star™ (DE3) cells and purified from the culture supernatant using the MagneHis™ Protein Purification System. The purified protein was used to immunize rabbits and produce an affinity-purified rabbit anti-goldfish granulin IgG for immunodetection. The purified protein was also added to goldfish primary kidney macrophage cultures to determine if granulin stimulates macrophage proliferation. (3567)

Notes: The authors devised an mRNA display system to generate a peptide library with multiple nonnatural amino acids incorporated into the proteins, an important feature of peptide libraries for successful drug discovery. An mRNA with 3 four-base codons at a random position was used as a template in an in vitro translation system in the presence of charged tRNAs carrying four-base codons. In vitro translations were performed using 3.6 × 10¹³ molecules of mRNA template and the E. coli S30 Extract System. The mRNA template contained a T7 tag sequence, so the translation products could be detected using an anti-T7 tag antibody and the Anti-Mouse IgG (H+L), AP Conjugate. The mRNA-displayed peptides also incorporated a polyhistidine tag so that they could be purified using the MagneHis™ Ni-Particles. After selecting for the desired protein characteristic, the mRNA portion of the mRNA-displayed peptides was reverse transcribed and quantitated by real-time PCR. PCR products were cloned into the pGEM^®-T Vector prior to sequencing. (3651)

Notes: These authors used a two-step process to purify a spectrum of ubiquitylated proteins from yeast expressing polyhistidine-tagged ubiquitin. First, two polyubiquitin-binding proteins were fused to glutathione-S-transferase purification tags (GST-Rad23 and GST-Dsk2), expressed in bacteria and purified by glutathione-Sepharose resin. A cleared yeast lysate was added to the GST-coupled proteins, mixed and washed. Elution of bound proteins was performed using a urea buffer (8M urea, 100mM NaH₂PO₄, 10mM Tris-HCl, pH 8.0). The eluate was then mixed with 125µl MagneHis™ Ni-Particles previously washed in the urea buffer. After incubation, the MagneHis™ particles were stringently rinsed with urea buffer + 0.5% Triton^® X-100. To generate peptides for MS-based sequencing, proteolytic digests were performed directly on the beads. The enzymes used were endoproteinase Lys-C (incubated for 5 hours) and trypsin (incubated for 16 hours). After digestion, the protein sample was analyzed by mass spectrometry (ESI-MS). (3286)

Notes: In this study, the researchers used the MagneHis™ Protein Purification System to purify recombinant, histidine-tagged Tribolium castaneum (red grain beetle) esterase from Pichia pastoris. The T. castaneum esterase gene, termed TCE, was cloned into pGAPZα A, pGAPZ B, and pPICZ B vectors and P. pastoris cultures transformed with each vector were analyzed for esterase activity. TCE yields varying from 7-80mg/L were obtained from P. pastoris cultures containing the above constructs using the MagneHis™ Protein Purification System. Specific activities of histidine-tagged TCE ranged from 4.5 to 5.7 U/mg. (3297)

Notes: These authors studied the activity of oxygen-independent coproporphyrinogen III oxidase HemN, an enzyme involved in converting coproporphyrinogen III to protoporphyrinogen IX in heme and chlorophyll biosynthesis. The activity assay for HemN requires protoporphyrinogen IX oxidase to convert the end products of the HemN reaction to a detectable form. Recombinant protoporphyrinogen IX oxidase was expressed as a polyhistidine-tagged protein in E. coli strain BL21-Codon-Plus(DE3)-RIL and purified using the MagneHis™ Protein Purification System. (3569)

Notes: Researchers compared MagneHis™ Ni-Particles to other vendors’ his tag protein purification systems in a model co-precipitation system with various bait proteins and a Shewanella oneidensis degradosome. Peptides from the S. oneidensis degradosome that co-purified with the bait proteins were analyzed by SEQUEST analysis. Bait proteins included E. coli polynucleotide phosphorylase, (PNP), RNase E and the RNA helicase. The authors describe the use of the MagneHis™ Ni-Particles in a simple and efficient system that can be automated for screening purposes. The authors also discussed optimizing the elution conditions, amount of bait protein and wash steps. (3280)

Notes: These authors characterized metabolic pathways in Borrelia burgdorferi, the causative agent of Lyme disease, focusing on the 5´-methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) and the autoinducer-2 production protein LuxS. Recombinant LuxS and Pfs proteins from both B. burgdorferi and E. coli were expressed as polyhistidine-tagged proteins in BL(21)DE3pLysE and purified using the MagneHis™ Protein Purification System. The purified proteins were then used in enzyme activity assays. The E. coli LuxS and Pfs proteins were used as positive controls for enzyme activity. (3566)

Notes: In this study, the fucosidase domain of the Bifidobacterium bifidum 1,2-alpha-L-fucosidase was purified as a carboxy-terminal fusion to hexahistidine. The fucosidase domain was cloned into an inducible T7 expression vector and transformed into the bacterial strain BL21(DE3). The expressed protein was then purified using the MagneHis Protein Purification System. The protein was eluted with a gradient of 0-1M NaCl in 10mM sodium phosphate buffer (pH 6.5). (3170)

Notes: Researchers used MagneHis™ Ni-Particles to purify his-tagged peptidoglycan recognition protein-1 (PGRP1 and PGRP2) that had been excreted into medium supernatants. The his-tagged proteins were created by making fusion-protein expression vectors from isolated H. diomphalia larvae cDNA and the pMT/Bip/V5-His vector (Invitrogen). The construct was then stably transfected into Drosophila Schneider S2 cells, and the medium was monitored for secreted protein by Western blot analysis. (2837)

Notes: MagneHis™ Ni-Particles were used to enrich for histidine-tagged T7 RNA Polymerase DNA bound to templates with T7 promoters. The templates contained various platinum adducts that stalled or stopped in vitro transcription reactions with T7 polymerase. (2818)