|
| 1. |
Muik, M., Frischauf, I., Derler, I., Fahrner, M., Bergsmann, J., Eder, P., Schindl, R., Hesch, C., Polzinger, B., Fritsch, R., Kahr, H., Madl, J., Gruber, H., Groschner, K. and Romanin, C.
(2008)
Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation.
J. Biol. Chem.
283
,
8014–8022
.
|
| |
Notes:
The authors performed protein pull-down assays to characterize the interaction of ORAI1 and STIM1, two protein components of the calcium-release calcium current. His6-STIM1 C terminus and ORAI1 were synthesized using the TNT® Coupled Reticulocyte Lysate System in the presence of 35S, and His6-STIM1 C terminus was immobilized using MagZ™ Binding Particles. An aliquot of the TNT® reaction expressing ORAI1 was added to the particles, and proteins were washed, eluted using increasing concentrations of imidazole (10–40mM) and analyzed by SDS-PAGE. In a second set of pull-down assays, His6-STIM1 C terminus was used to pull down ORA1 N- and C-terminal fragments expressed as GST fusion proteins. The His6-STIM1 C terminus protein was purified from transiently tranfected HEK293 cells using the MagneHis™ Protein Purification System.
(0003781) |
| |
 |
| |
Products: MagneHis™ Protein Purification System | MagZ™ Protein Purification System | TNT® T7 Coupled Reticulocyte Lysate System |
| 2. |
Mie, M., Shimizu, S., Takahashi, F. and Kobatake, E.
(2008)
Selection of mRNA 5´-untranslated region sequence with high translation efficiency through ribosome display.
Biochem. Biophys. Res. Commun.
373
,
48–52
.
|
| |
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.
(0003963) |
| |
|
| |
Products: Renilla Luciferase Assay System | Flexi® Rabbit Reticulocyte Lysate System | MagneHis™ Ni-Particles | TNT® T7 Coupled Reticulocyte Lysate System |
| 3. |
Cheung, Q.C., Turner, P.V., Song, C., Wu, D., Cai, H.Y., MacInnes, J.I. and Li, J.
(2008)
Enhanced resistance to bacterial infection in protegrin-1 transgenic mice.
Antimicrob. Agents Chemother.
52
,
1812–9
.
|
| |
Notes:
One potential source of antibiotic-resistant bacteria is food-producing animals. The authors examined the ability of protegrin-1 (PG-1), an antimicrobial peptide, to protect wildtype and transgenic mice expressing PG-1 against bacterial infection. As part of the cloning strategy to produce the PG-1 expression construct, the authors amplified and cloned full-length PG-1 into the pGEM®-T Easy Vector. To test the bactericidal activity of PG-1 expressed in transgenic mice, radial diffusion assays were performed, in which test samples were added to a well containing E. coli and the clear antibacterial zone was measured. Two of the test samples were neutrophil secretions from the PG-1 transgenic mice and purified polyhistidine-tagged PG-1 protein, purified using the MagneHis™ Protein Purification System.
(0003896) |
| |
|
| |
Products: MagneHis™ Protein Purification System | pGEM®-T Easy Vector System I |
| 4. |
Hanington, P.C., Brennan, L.J., Belosevic, M. and Keddie, B.A.
(2008)
Molecular and functional characterization of granulin-like molecules of insects.
Insect Biochem. Mol. Biol.
38
,
596–603
.
|
| |
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.
(0003964) |
| |
|
| |
Products: MagneHis™ Ni-Particles |
| 5. |
Schäfer, P., Cymerman, I.A., Bujnicki, J.M. and Meiss, G.
(2007)
Human lysosomal DNase IIα contains two requisite PLD-signature (HxK) motifs: evidence for a pseudodimeric structure of the active enzyme species.
Protein Sci.
16
,
82–91
.
|
| |
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.
(0003786) |
| |
|
| |
Products: MagneHis™ Ni-Particles | pCI Mammalian Expression Vector | TransFast™ Transfection Reagent |
| 6. |
Mi, Z., Oliver, T., Guo, H., Gao, C. and Kuo, P.C.
(2007)
Thrombin-cleaved COOH(-) terminal osteopontin peptide binds with cyclophilin C to CD147 in murine breast cancer cells.
Cancer Res.
67
,
4088–97
.
|
| |
Notes:
The authors investigated the role of short COOH-terminal osteopontin (SC-OPN), which is a product of thrombin cleavage of osteopontin, in tumor metastasis. The authors expressed SC-OPN as a fusion with a cyan-shifted variant of green fluorescent protein (SC-OPN-CFP)and cyclophilin C, a marker of metastatic function, as a fusion with the yellow-shifted variant (CyC-YFP). The fusion proteins were expressed with a His tag, and proteins were purified using the MagneHis™ Protein Purification System. Purifed SC-OPN-CFP and CyC-YFP were incubated with 4T07 cells and the cells were analyzed by flow cytometry and confocal microscopy to determine whether the proteins bound to the CD147 cell surface glycoprotein. In addition full-length OPN, truncated forms of OPN, and osteopontin with a mutated thrombin site (Mu-OPN) were expressed as his-tagged proteins and purified from COS7 cells using the MagneHis™ Protein Purification System. These purified proteins were added to the mouse mammary tumor cell line 4T07, and cell migration and invasiveness were measured to determine the effect on metastatic activity.
(0003787) |
| |
|
| |
Products: MagneHis™ Protein Purification System |
| 7. |
Muranaka, N., Hohsaka, T. and Sisido, M.
(2006)
Four-base codon mediated mRNA display to construct peptide libraries that contain multiple nonnatural amino acids.
Nucleic Acids Res.
34
,
e7
.
|
| |
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.
(0003651) |
| |
|
| |
Products: E. coli S30 Extract System for Linear Templates | Anti-Mouse IgG (H+L), AP Conjugate | MagneHis™ Ni-Particles | MagneHis™ Protein Purification System | pGEM®-T Vector System I | pGEM®-T Vector System II |
| 8. |
Hanington, P.C., Barreda, D.R. and Belosevic, M.
(2006)
A novel hematopoietic granulin induces proliferation of goldfish (Carassius auratus L.) macrophages.
J. Biol. Chem.
281
,
9963–9970
.
|
| |
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.
(0003567) |
| |
|
| |
Products: MagneHis™ Ni-Particles | MagneHis™ Protein Purification System |
| 9. |
Nakase, Y., Fukuda, K., Chikashige, Y., Tsutsumi, C., Morita, D., Kawamoto, S., Ohnuki, M., Hiraoka, Y. and Matsumoto, T.
(2006)
A defect in protein farnesylation suppresses a loss of Schizosaccharomyces pombe tsc2+, a homolog of the human gene predisposing to tuberous sclerosis complex.
Genetics
173
,
569–578
.
|
| |
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.
(0003568) |
| |
|
| |
Products: MagneHis™ Ni-Particles | MagneHis™ Protein Purification System |
| 10. |
Lin, C.T., Moore, P.A., Auberry, D.L., Landorf, E.V., Peppler, T., Victry, K.D., Collart, F.R. and Kery, V.
(2006)
Automated purification of recombinant proteins: combining high-throughput with high yield.
Protein Expr. Purif.
47
,
16–24
.
|
| |
Notes:
The authors compared two methods to purify a subset of 236 hypothetical hexahistidine-tagged Shewanella oneidensis proteins: a large-scale, lower-throughput filtration separation protocol using Ni-NTA Superflow columns (Qiagen) and a lower-scale but higher-throughput magnetic bead-based purification protocol using MagneHis™ Ni-Particles. They examined several factors that can affect yield and efficiency of protein binding to these two matrices, including steric factors, protein size, amount of cell lysate and cell lysis protocol. They concluded that both matrices seem to have similar protein-binding capacities, and the larger-scale and lower-scale methods resulted in 8.7µg/OD600 and 8.8µg/OD600, respectively. When examining native proteins of similar sizes, they found binding differences between monomeric and oligomeric forms, most likely due to steric hindrances around the polyhistidine tag. However, they found no correlation between protein yield and protein size (as measured in kDa or as Stokes radius). The authors calculated that the maximum yield was approximately 200µg of protein from a lysate load of 30 OD600 and observed that some proteins are difficult to elute from the MagneHis™ Ni-Particles. Inefficient elution can affect yield. About 30% of proteins were purified to >90% homogeneity and about 40% to >80% homogeneity using MagneHis™ Ni-Particles. Fourteen of the proteins were not expressed in sufficient quantity for protein purification. Finally, the authors concluded that the automated filtration process does not save much time or labor compared to the manual filtration process, and the lower-scale MagneHis™ protocol is efficient with minimal error rate. The authors used the Biomek® FX automated workstation to process 96-well plates of E. coli cultures expressing the various S. oneidensis proteins.
(0003848) |
| |
|
| |
Products: MagneHis™ Protein Purification System |
| 11. |
Markillie, L.M., Lin, C.T., Adkins, J.N., Auberry, D.L., Hill, E.A., Hooker, B.S., Moore, P.A., Moore, R.J., Shi, L., Wiley, H.S., and Kery, V.
(2005)
Simple protein complex purification and identification method for high-throughput mapping of protein interaction networks.
J. Proteome Res.
4
,
268-274
.
|
| |
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.
(0003280) |
| |
|
| |
Products: MagneHis™ Ni-Particles | MagneHis™ Protein Purification System |
| 12. |
Delroisse, J.M., Dannau, M., Gilsoul, J.J., El Mejdoub, T., Destain, J., Portetelle, D., Thonart, P., Haubruge, E., and Vandenbol, M.
(2005)
Expression of a synthetic gene encoding a Tribolium castaneum carboxylesterase in Pichia pastoris.
Prot. Express. and Purif.
42
,
286–294
.
|
| |
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.
(0003297) |
| |
|
| |
Products: MagneHis™ Ni-Particles | MagneHis™ Protein Purification System |
| 13. |
Babb, K., von Lackum, K., Wattier, R.L., Riley, S.P. and Stevenson, B.
(2005)
Synthesis of autoinducer 2 by the Lyme disease spirochete, Borrelia burgdorferi.
J. Bacteriol.
187
,
3079–3087
.
|
| |
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.
(0003566) |
| |
|
| |
Products: MagneHis™ Ni-Particles | MagneHis™ Protein Purification System |
| 14. |
Layer, G., Grage, K., Teschner, T., Schünemann, V., Breckau, D., Masoumi, A., Jahn, M., Heathcote, P., Trautwein, A.X. and Jahn, D.
(2005)
Radical S-adenosylmethionine enzyme coproporphyrinogen III oxidase HemN.
J. Biol. Chem.
280
,
29038–46
.
|
| |
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.
(0003569) |
| |
|
| |
Products: MagneHis™ Ni-Particles | MagneHis™ Protein Purification System |
| 15. |
Mayor, T., Lipford, J.R., Graumann, J., Smith, G.T. and Deshaies, R.J.
(2005)
Analysis of polyubiquitin conjugates reveals that the rpn10 substrate receptor contributes to the turnover of multiple proteasome targets.
Mol. Cell. Proteomics
4
,
741–51
.
|
| |
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).
(0003286) |
| |
|
| |
Products: MagneHis™ Ni-Particles |
| 16. |
Katayama, T., Sakuma, A., Kimura, T., Makimura, Y., Hiratake, J., Sakata, K., Yamanoi, T., Kumagai, H. and Yamamoto, K.
(2004)
Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-L-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95)
J. Bacteriol.
186(15)
,
4885-4893
.
|
| |
Notes:
In this study, the fucosidase domain of the Bifidobacterium bifidum 1,2-alpha-L-fucosidase was purified as a carboxy-terminal fusion to hexahistadine. 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).
(0003170) |
| |
|
| |
Products: MagneHis™ Ni-Particles | MagneHis™ Protein Purification System |
| 17. |
Lee, M.H., Osaki, T., Lee, J.Y., Baek, M.J., Zhang, R., Park, J.W., Kawabata, S.I., Soderhall, K., and Lee, B.L.
(2004)
Peptidoglycan recognition proteins involved in 1,3-beta-D-glucan-dependent prophenoloxidase activation system of insect.
J. Biol. Chem.
279(5)
,
3218–3227
.
|
| |
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.
(0002837) |
| |
|
| |
Products: MagneHis™ Ni-Particles |
| 18. |
Jung, Y. and Lippard, S.J.
(2003)
Multiple states of stalled T7 RNA polymerase at DNA lesions generated by platinum anticancer agents.
J. Biol. Chem.
278
,
52084-52092
.
|
| |
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.
(0002818) |
| |
|
| |
Products: MagneHis™ Ni-Particles |