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Epitope Tag (epitope + tag)
Selected AbstractsConstitutive oligomerization of human D2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cellsFEBS JOURNAL, Issue 19 2003Analysis using co-immunoprecipitation, time-resolved fluorescence resonance energy transfer Human D2Long (D2L) and D2Short (D2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [3H]Spiperone labelled D2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D2L or D2S receptors, with a pKd value of , 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D2L and D2S receptors in Sf9 cells. When the FLAG-tagged D2S and HIV-tagged D2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D2 receptors. In both cases, constitutive homo-oligomers were revealed for D2L and D2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D2S receptors. The D2 receptor ligands dopamine, R -(,)propylnorapomorphine, and raclopride did not affect oligomerization of D2L and D2S in Sf9 and HEK293 cells. Human D2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D2 oligomerization in these cells is not regulated by ligands. [source] The structure of the TGF-, latency associated peptide region determines the ability of the proprotein convertase furin to cleave TGF-,sJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2008Makoto Kusakabe Abstract The TGF-, family members are generated as latent pre-pro-polypeptides. The active mature peptides are cleaved from the latent forms by cellular proteases. TGF-,1, for instance, is predominantly processed by a substilisin-like proprotein convertase, furin. TGF-,2 has a consensus cleavage site for furin and therefore has been presumed to be cleaved by furin. However, TGF-,2 is often secreted as the latent form, which appears to be inconsistent with its postulated sensitivity to furin. We report here that both the regular (short) form of TGF-,2 and its spliced variant with an additional exon (long form) are insensitive to furin. NIH 3T3 and CHO cells were transfected with expression vectors containing the short or long form of TGF-,2 or a chimeric TGF-, consisting of the TGF-,1 LAP region, the TGF-,2 cleavage site and the TGF-,2 mature peptide. The constructs included a c- myc epitope tag in the N-terminal region of the mature peptide. The TGF-,s produced by the transfected cells were analyzed with Western blots and immunocytochemistry. The intracellular proteins harvested from these cells were incubated with furin. Furin only inefficiently cleaved both the long and short forms of TGF-,2, but efficiently processed the chimeric TGF-,. This indicates that the insensitivity of both forms of TGF-,2 to furin is a consequence of the tertiary structure of their LAP regions rather than their cleavage site. This differential processing of TGF-,1 and -,2 may be part of the mechanism that generates isoform-specific functions of the TGF-,s. J. Cell. Biochem. 103: 311,320, 2008. © 2007 Wiley-Liss, Inc. [source] Transgenic mice exhibiting oligodendrocyte-specific expression of a mutant protein tyrosine phosphatase epsilonJOURNAL OF NEUROCHEMISTRY, Issue 2002N. Muja Reversible tyrosine phosphorylation is integral to oligodendrocyte differentiation, and one participant of the phosphorylation cycle, PTP,, is induced in developing oligodendrocytes (Ranjan and Hudson, 1996). To define the role of PTP,, we generated mice expressing a catalytically inactive, hemagglutinin-epitope tagged PTP, (HA-PTP ,) from the 2,,3,-cyclic nucleotide 3,-phosphodiesterase (CNP) promoter. By competing with endogenous, normal PTP, for substrate binding, HA-PTP, would behave in a dominant negative fashion when overexpressed in these mice. Transgene mRNA peaks at postnatal day 21, coincident with the maximal expression of myelin protein mRNAs. Immunohistochemical analyses using antibodies against the HA epitope tag demonstrate that HA-PTP, is expressed in oligodendrocytes, but not in astrocytes and neurons. HA immunoreactivity was present in all myelinated brain structures including the corpus callosum, anterior commissure, and fornix, as well as in the subcortical, cerebellar, and spinal cord white matter. Gross differences in myelination or oligodendrocyte cell density in these brain regions were not detected using antibodies against CNP, myelin basic protein, and an oligodendrocyte marker, CC1. However, by EM axons of the optic nerve appear smaller and less extensively myelinated in transgenic mice than in wild-type littermates. Studies are underway to determine the functional effects of transgene expression on conduction velocity, on the profile of expressed genes, and on potential phosphorylated protein targets of PTP,. [source] Crystallization and crystal-packing studies of Chlorella virus deoxyuridine triphosphataseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Kohei Homma The 141-amino-acid deoxyuridine triphosphatase (dUTPase) from the algal Chlorella virus IL-3A and its Glu81Ser/Thr84Arg-mutant derivative Mu-22 were crystallized using the hanging-drop vapor-diffusion method at 298,K with polyethylene glycol as the precipitant. An apo IL-3A dUTPase with an amino-terminal T7 epitope tag and a carboxy-terminal histidine tag yielded cubic P213 crystals with unit-cell parameter a = 106.65,Å. In the presence of dUDP, the enzyme produced thin stacked orthorhombic P222 crystals with unit-cell parameters a = 81.0, b = 96.2, c = 132.8,Å. T7-histidine-tagged Mu-22 dUTPase formed thin stacked rectangular crystals. Amino-terminal histidine-tagged dUTPases did not crystallize but formed aggregates. Glycyl-seryl-tagged dUTPases yielded cubic P213 IL-3A crystals with unit-cell parameter a = 105.68,Å and hexagonal P63 Mu-22 crystals with unit-cell parameters a = 132.07, c = 53.45,Å, , = 120°. Owing to the Thr84Arg mutation, Mu-22 dUTPase had different monomer-to-monomer interactions to those of IL-3A dUTPase. [source] A PCR-based strategy to generate yeast strains expressing endogenous levels of amino-terminal epitope-tagged proteinsBIOTECHNOLOGY JOURNAL, Issue 4 2008Keith R. Booher Abstract An epitope tag introduced to a gene of interest (GOI) greatly increases the ease of studying cellular proteins. Rapid PCR-based strategies for epitope tagging a protein's C-terminus at its native gene locus are widely used in yeast. C-terminal epitope tagging is not suitable for all proteins, however. Epitope tags fused to the C-terminus can interfere with function of some proteins or can even be removed by C-terminal protein processing. To overcome such problems, proteins can be tagged with epitopes at their amino-termini, but generating yeast strains expressing N-terminal epitope tagged genes under control of the endogenous promoter at the native locus is comparatively more difficult. Strategies to introduce N-terminal epitope tags have been reported previously but often introduce additional sequences other than the epitope tag into the genome. Furthermore, N-terminal tagging of essential genes by current methods requires formation of diploid strains followed by tetrad dissection or expression of an additional copy of the GOI from a plasmid. The strategies described here provide a quick, facile means of epitope tagging the N-terminus of both essential and nonessential genes in a two-step PCR-based procedure. The procedure has the significant advantage of leaving tagged genes under the control of their endogenous promoters, and no additional sequences other than the epitope tag encoding nucleotides are inserted into the genome. [source] | |||||||||||||