2014
DOI: 10.1002/0471141755.ph0211s67
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Arrestin Expression in E. coli and Purification

Abstract: Purified arrestin proteins are necessary for biochemical, biophysical, and crystallographic studies of these versatile regulators of cell signaling. Here we describe a basic protocol for expression in E. coli and purification of tag-free wild type and mutant arrestins. The method includes ammonium sulfate precipitation of arrestins from cell lysates, followed by heparin-Sepharose chromatography. Depending on the arrestin type and/or mutations, this step is followed by Q-Sepharose or SP-Sepharose chromatography… Show more

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Cited by 23 publications
(27 citation statements)
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“…Arrestin-3 (1-393) was expressed and purified as described (91)(92)(93)(94)(95) followed by an additional purification step on a Superdex S200 Increase 10/300 GL column (GE Life Sciences, Pittsburgh, PA) equilibrated in 20 mM MOPS, pH 7.5, 150 mM NaCl, and 2 mM TCEP. Protein purity was assessed by SDS-PAGE with Coomassie staining.…”
Section: Arrestin-3-(1-393) Expression and Purificationmentioning
confidence: 99%
“…Arrestin-3 (1-393) was expressed and purified as described (91)(92)(93)(94)(95) followed by an additional purification step on a Superdex S200 Increase 10/300 GL column (GE Life Sciences, Pittsburgh, PA) equilibrated in 20 mM MOPS, pH 7.5, 150 mM NaCl, and 2 mM TCEP. Protein purity was assessed by SDS-PAGE with Coomassie staining.…”
Section: Arrestin-3-(1-393) Expression and Purificationmentioning
confidence: 99%
“…WT visual arrestin-1 from different species (bovine, mouse, and human) successfully folds even in E. coli , which has fewer chaperones than eukaryotic cells and can be purified from expressing bacteria in large quantities. 25 , 26 Therefore, next we compared the expression of human arrestin-1 C147F mutant with the WT human and bovine proteins in E. coli , as well as the soluble fraction of these proteins ( Fig. 2 C).…”
Section: Resultsmentioning
confidence: 99%
“… 25 Test expression in 3 mL bacterial culture, cell lysis, and separation of soluble protein from cell debris were performed, as described. 26 In vitro transcription, translation, preparation of phosphorylated and unphosphorylated rhodopsin were performed as described recently. 27 , 28 …”
Section: Methodsmentioning
confidence: 99%
See 1 more Smart Citation
“…To assess whether the differences in downstream signaling of TRV027 and SII could result from differential conformational rearrangements of Ī²-arrestin as previously shown for other AT 1 receptor ligands (Shukla et al, 2008 ; Zimmerman et al, 2012 ; Gurevich and Gurevich, 2014 ), we used a BRET-based biosensor that monitors the conformational rearrangement of Ī²-arrestins by measuring intra-molecular BRET between RLucII and GFP 10 fused to the N- and C-termini of Ī²-arrestins respectively (RlucII-Ī²arrestin-GFP 10 ) (Charest et al, 2005 ). Using this approach, the amplitude of BRET changes provides an indirect reflection of the conformational rearrangement of Ī²-arrestin upon its recruitment to the receptor promoted by a given ligand.…”
Section: Resultsmentioning
confidence: 99%