Arrestin expression in E. coli and purification.

Vishnivetskiy SA, Zhan X, Chen Q, Iverson TM, Gurevich VV
Curr Protoc Pharmacol. 2014 67: Unit 2.11.1-19

PMID: 25446290 · PMCID: PMC4260927 · DOI:10.1002/0471141755.ph0211s67

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.

Copyright © 2014 John Wiley & Sons, Inc.

MeSH Terms (4)

Arrestins Chromatography, Agarose Escherichia coli Escherichia coli Proteins

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