Notes: Fructokinase-like proteins (FLNs) are components of the plastid-encoded RNA polymerase (PEP), which is responsible for transcribing chloroplast genes. Here, a novel heat-stress phenotype in rice is linked to a mutation in FLNs. Knockdowns and knockouts of fln1 show inhibition of chloroplast biogenesis. FLN1 and 2 were purified using the MagneGST™ Pull-Down System, and a direct interaction was validated with an in vitro pull-down assay. (5102)

Notes: The authors investigated the role of the ubiquitin E3 ligase PDLIM2 in the degradation of signal tranducer and activator of transcription 1 (STAT1). They showed that activation of PDLIM2 and subsequent STAT1 ubiquitination require protein kinase C-mediated phoshorylation of PDLIM2 on serine 137. Polyhistidine-tagged PDLIM2 and polyhistidine-tagged mutants PDLIM2-S137A and PDLIM2-S137D were purified using the MagneHis™ Protein Purification System for use in in vitro phosphorylation and ubiquitination assays. One mechanism used to assess levels of STAT1 was a reporter assay using RAW264.7 cells transfected with a pGL3-based construct containing a interferon γ-activated sequence (GAS) upstream of the firefly luciferase reporter gene. Expression of wildtype PDLIM2, but not the mutant forms, resulted in much lower levels of STAT1 protein, and thus lower luciferase activity, when cells were challenged with lipopolysaccharide. The pRL-TK Vector was used to normalize for transfection efficiency. Luciferase assays were performed using the Dual Luciferase^® Reporter Assay System and Reporter Lysis Buffer. (4152)

Notes: The authors investigated the expression of genes encoding histone-like (H-NS) proteins from the self-transmissible pCAR1 plasmid and Pseudomonas putida KT2440 genome, as well as the interaction of H-NS family members in vitro. Gene expression was quantified using quantitative RT-PCR and RNA templates that were treated with RQ1 RNase-Free DNase to degrade contaminating DNA. Interactions between Pmr and other H-NS proteins were monitored using pull-down assays. His-tagged Pmr was expressed in E. coli, purified and used as bait for FLAG-tagged H-NS family proteins. Protein purification of His-tagged proteins was performed using the MagneHis™ Protein Purification System. (4119)

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 transfected HEK293 cells using the MagneHis™ Protein Purification System. (3781)

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. (3896)

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. Purified 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. (3787)

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 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. (3848)

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: 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)