title: 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. Described herein is a basic protocol for arrestin expression in E. coli and purification of the 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, the next step is Q-Sepharose or SP-Sepharose chromatography. In many cases the nonbinding column is used as a filter to bind contaminants without retaining arrestin. In some cases both chromatographic steps must be performed sequentially to achieve high purity. Purified arrestins can be concentrated up to 10 mg/ml, remain fully functional, and withstand several cycles of freezing and thawing, provided that overall salt concentration is maintained at or above physiological levels.
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, Tennessee.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Vishnivetskiy
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, Tennessee.
authors.forename: Xuanzhi
authors.initials: X
authors.lastname: Zhan
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, Tennessee.
authors.forename: Qiuyan
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, Tennessee.
authors.forename: Tina M
authors.initials: TM
authors.lastname: Iverson
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, Tennessee.
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Gurevich
mesh_terms: D019390:Arrestins / Q000096:biosynthesis* / Q000302:isolation & purification; D002847:Chromatography, Agarose; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D019390:Arrestins; D029968:Escherichia coli Proteins
keywords: arrestin; chromatography; expression; purification; recombinant