Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.
TL;DR: Plasmid expression vectors have been constructed that direct the synthesis of foreign polypeptides in Escherichia coli as fusions with the C terminus of Sj26, a 26-kDa glutathione S-transferase (GST; EC 2.5.1.18) encoded by the parasitic helminth Schistosoma japonicum.
About: This article is published in Gene.The article was published on 1988-07-15. It has received 6003 citations till now. The article focuses on the topics: Fusion protein & Expression vector.
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TL;DR: Molecular genetic and biochemical studies described here suggest that, as in the case of growth factor receptors of higher eukaryotic cells, Ire1p oligomerizes in response to the accumulation of unfolded proteins in the ER and is phosphorylated in trans by otherIre1p molecules as a result of oligomerization.
Abstract: The transmembrane kinase Ire1p is required for activation of the unfolded protein response (UPR), the increase in transcription of genes encoding endoplasmic reticulum (ER) resident proteins that occurs in response to the accumulation of unfolded proteins in the ER. Ire1p spans the ER membrane (or the nuclear membrane with which the ER is continuous), with its kinase domain localized in the cytoplasm or in the nucleus. Consistent with this arrangement, it has been proposed that Ire1p senses the accumulation of unfolded proteins in the ER and transmits the signal across the membrane toward the transcription machinery, possibly by phosphorylating downstream components of the UPR pathway. Molecular genetic and biochemical studies described here suggest that, as in the case of growth factor receptors of higher eukaryotic cells, Ire1p oligomerizes in response to the accumulation of unfolded proteins in the ER and is phosphorylated in trans by other Ire1p molecules as a result of oligomerization. In addition to its kinase domain, a C-terminal tail domain of Ire1p is required for induction of the UPR. The role of the tail is probably to bind other proteins that transmit the unfolded protein signal to the nucleus.
12,185 citations
TL;DR: It is demonstrated that the rat microtubule‐associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing.
Abstract: Little is known about the protein constituents of autophagosome membranes in mammalian cells. Here we demonstrate that the rat microtubule-associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing. Two forms of LC3, called LC3-I and -II, were produced post-translationally in various cells. LC3-I is cytosolic, whereas LC3-II is membrane bound. The autophagic vacuole fraction prepared from starved rat liver was enriched with LC3-II. Immunoelectron microscopy on LC3 revealed specific labelling of autophagosome membranes in addition to the cytoplasmic labelling. LC3-II was present both inside and outside of autophagosomes. Mutational analyses suggest that LC3-I is formed by the removal of the C-terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3-I into LC3-II. The amount of LC3-II is correlated with the extent of autophagosome formation. LC3-II is the first mammalian protein identified that specifically associates with autophagosome membranes.
6,244 citations
TL;DR: A new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications that should further facilitate the rapid analysis of gene function in S. cerevisiae.
Abstract: An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications Using as selectable marker the S cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5 + or Escherichia coli kan r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging) Because of the modular nature of the plasmids, they allow eYcient and economical use of a small number of PCR primers for a wide variety of gene manipulations Thus, these plasmids should further facilitate the rapid analysis of gene function in S cerevisiae ? 1998 John Wiley & Sons, Ltd
5,301 citations
Cites background or methods from "Single-step purification of polypep..."
...1 15X Myc (containing sequences encoding 15 tandem repeats of the Myc epitope) was kindly provided by O. Mondesert and P. Russell; plasmid pGEX-2T (Smith and Johnson, 1988) contains the Yeast 14, 953–961 (1998) ?...
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...…both the localization of proteins by immunofluorescence (Bi and Pringle, 1996; Longtine et al., 1998) and the rapid, one-step biochemical purification of both the tagged protein and associated proteins using glutathione-conjugated agarose beads (Smith and Johnson, 1988; Ausubel et al., 1995)....
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...Yeast 14, 953–961 (1998) Additional Modules for Versatile and Economical PCR-based Gene Deletion and Modification in...
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TL;DR: Rho, a ras-related GTP-binding protein, rapidly stimulated stress fiber and focal adhesion formation when microinjected into serum-starved Swiss 3T3 cells, implying that rho is essential specifically for the coordinated assembly of focal adhesions and stress fibers induced by growth factors.
4,365 citations
TL;DR: The results suggest that calcineurin is involved in a common step associated with T cell receptor and IgE receptor signaling pathways and that cyclophilin and FKBP mediate the actions of CsA and Fk506 by forming drug-dependent complexes with and altering the activity of calcineURin-calmodulin.
3,968 citations
Cites methods from "Single-step purification of polypep..."
...A Common Set of Proteins Bind to Cyclophilin-CsA and FKBP-FK506, but Not Cyclophilin, FKBP, CsA, FK506, Rapamycin, or FKBP-Rapamycin A chimeric gene encoding a glutathione S-transferase-FKBP12 fusion protein (GFK) was constructed by fusing the cDNA encoding FKBP12 to that encoding the carboxyl terminus of glutathione S-transferase ( Smith and Johnson, 1988 )....
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...Sepharose as described previously ( Smith and Johnson, 1988 )....
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References
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TL;DR: New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors and mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences.
14,954 citations
TL;DR: Under the new conditions there is direct proportionality between absorbance at 650 nm and weight of protein within the range 15–110 μg.
5,177 citations
TL;DR: Pulse-chase experiments in wild-type and mutant phage-infected cells provide evidence that the following particles called prohead I, II and III are successive precursors to the mature heads as discussed by the authors.
3,697 citations
01 Apr 1985
TL;DR: A method for obtaining sequence information directly from plasmid DNA is presented, and the advantages include speed, simplicity, avoidance of additional cloning steps into single-stranded phage M13 vectors, and hence applicability to sequencing large numbers of samples.
Abstract: A method for obtaining sequence information directly from plasmid DNA is presented. The procedure involves the rapid preparation of clean supercoiled plasmid DNA from small bacterial cultures, its complete denaturation by alkali, and sequence determination using oligodeoxyribonucleotide-primed enzymatic DNA synthesis in the presence of dideoxynucleoside triphosphates. The advantages of the method include speed, simplicity, avoidance of additional cloning steps into single-stranded phage M13 vectors, and hence applicability to sequencing large numbers of samples.
2,184 citations
TL;DR: A sensitive and general technique has been devised for the dual purposes of cloning genes by using antibodies as probes and isolating unknown proteins encoded by cloned DNA using an expression vector that permits insertion of foreign DNA into the beta-galactosidase structural gene lacZ and promotes synthesis of hybrid proteins.
Abstract: A sensitive and general technique has been devised for the dual purposes of cloning genes by using antibodies as probes and isolating unknown proteins encoded by cloned DNA. The method uses an expression vector, lambda gt11 (lac5 nin5 cI857 S100), that permits insertion of foreign DNA into the beta-galactosidase structural gene lacZ and promotes synthesis of hybrid proteins. Efficient screening of antigen-producing clones in lambda gt11 recombinant cDNA libraries is achieved through lysogeny of the phage library in hflA (high-frequency lysogeny) mutant cells of Escherichia coli; lysogens produce detectable quantities of antigen on induction, even when plated at high cell densities. The vector is also designed to facilitate the isolation of proteins specified by previously cloned gene sequences. Hybrid proteins encoded by recombinant phage accumulate in strains defective in protein degradation (lon mutants) in amounts amenable to large-scale purification. Antibodies produced against the portion of the hybrid encoded by foreign DNA could in turn be used to isolate the native polypeptide from eukaryotic cells.
1,998 citations