Study of GPCR–Protein Interactions Using Gel Overlay Assays and Glutathione-S-Transferase-Fusion Protein Pull-Downs

AbstractIn order to demonstrate that an existing zinc-finger protein can be simply modified to enhance DNA binding and sequence discrimination in both episomal and chromatin contexts using existing zinc-finger DNA recognition code data, and without recourse to phage display and selection strategies, we have examined the consequences of a single zinc-finger extension to a synthetic three-zinc-finger VP16 fusion protein, on transcriptional activation from model target promoters harbouring the zinc-finger binding sequences. We report a nearly 10-fold enhanced transcriptional activation by the four-zinc-finger VP16 fusion protein relative to the progenitor three-finger VP16 protein in transient assays and a greater than five-fold enhancement in stable reporter-gene expression assays. A marked decrease in transcriptional activation was evident for the four-zinc-finger derivative from mutated regulatory regions compared to the progenitor protein, as a result of recognition site-size extension. This discriminatory effect was shown to be protein concentration-dependent. These observations suggest that four-zinc-finger proteins are stable functional motifs that can be a significant improvement over the progenitor three-zinc-finger protein, both in terms of specificity and the ability to target transcriptional function to promoters, and that single zinc-finger extension can therefore have a significant impact on DNA zinc-finger protein interactions. This is a simple route for modifying or enhancing the binding properties of existing synthetic zinc-finger-based transcription factors and may be particularly suited for the modification of endogenous zinc-finger transcription factors for promoter biasing applications.

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Functional and Physical Interactions of the Herpes Simplex Virus Type 1 UL20 Membrane Protein with Glycoprotein K

Journal of Virology ◽

10.1128/jvi.00147-08 ◽

2008 ◽

Vol 82 (13) ◽

pp. 6310-6323 ◽

Cited By ~ 36

Author(s):

Timothy P. Foster ◽

Vladimir N. Chouljenko ◽

K. G. Kousoulas

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Fusion Protein ◽

Protein Interactions ◽

Herpes Simplex Virus Type ◽

Protein Protein Interactions ◽

Glycoprotein K ◽

Er Retention ◽

Simplex Virus

ABSTRACT Herpes simplex virus type 1 glycoprotein K (gK) and the UL20 protein (UL20p) are coordinately transported to the trans-Golgi network (TGN) and cell surfaces and are required for cytoplasmic virion envelopment at the TGN. In addition, cell surface expression of gK and UL20p is required for virus-induced cell fusion. Previously, confocal microscopy colocalization and intracellular transport experiments strongly suggested direct protein-protein interactions between gK and UL20p. Direct protein-protein interactions between gK and UL20p were demonstrated through reciprocal coimmunoprecipitation experiments, as well as with glutathione S-transferase (GST) pull-down experiments. A fusion protein consisting of the amino-terminal 66 amino acids of UL20p fused in-frame with GST was expressed in Escherichia coli and purified via glutathione column chromatography. Precipitation of GST-UL20p from mixtures of GST-UL20p fusion protein with cellular extracts containing gK specifically coprecipitated gK but not other viral glycoproteins. The purified UL20p-GST fusion protein reacted with all gK-associated protein species. It was concluded that the amino terminus of UL20p, most likely, interacted with gK domain III, which is predicted to lie intracellularly. UL20p-gK domain-specific interactions must serve important functions in the coordinate transport of UL20p and gK to the TGN, because retention of UL20p in the endoplasmic reticulum (ER) via the addition of an ER retention signal at the carboxyl terminus of UL20p forced the ER retention of gK and drastically inhibited intracellular virion envelopment and virus-induced cell fusion.

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Expression, purification, and characterization of the carboxyl-terminal region of the Na+/H+ exchanger

Biochemistry and Cell Biology ◽

10.1139/o98-081 ◽

1998 ◽

Vol 76 (5) ◽

pp. 837-842 ◽

Cited By ~ 2

Author(s):

Daniel Gebreselassie ◽

Krishna Rajarathnam ◽

Larry Fliegel

Keyword(s):

Circular Dichroism ◽

Membrane Protein ◽

Fusion Protein ◽

Gel Electrophoresis ◽

Inclusion Bodies ◽

Cytoplasmic Domain ◽

Random Coil ◽

Glutathione S Transferase ◽

Carboxyl Terminal ◽

Circular Dichroïsm

The Na+/H+ exchanger is a pH regulatory protein that is responsible for removal of excess intracellular protons in exchange for extracellular Na+. It is a plasma membrane protein with a large cytoplasmic carboxyl terminal domain that regulates activity of the membrane domain. We overexpressed and purified the cytoplasmic domain that was produced in Escherichia coli. This region (516-815 amino acids) was under control of the tac promoter from the plasmid pGEX-KG and was fused with glutathione S-transferase. Upon induction, the fusion protein was principally found in inclusion bodies. Purified inclusion bodies were solubilized and fractionated using preparative SDS polyacrylamide gel electrophoresis. To obtain free Na+/H+ exchanger protein the fusion protein was dialyzed against cleavage buffer and cleaved at the thrombin cleavage site between glutathione S-transferase and the Na+/H+ exchanger domain. Free Na+/H+ exchanger protein was obtained by rerunning the sample on preparative gel electrophoresis. The final yield of the purified protein was 2.15 mg protein/L of cell culture. After exhaustive dialysis the secondary structure of the purified protein was assessed using circular dichroism spectroscopy. The results indicated that the protein was 35% alpha-helix, 17% beta-turn, and 48% random coil. They suggest that the cytoplasmic domain is structured and some regions may be compact in nature.Key words: Na+/H+ exchanger, pH regulation, membrane protein, circular dichroism.

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