Expression of Untagged Arrestins in E. coli and Their Purification

Abstract

AbstractPurified arrestin proteins are necessary for biochemical, biophysical, and structural studies of these versatile regulators of cell signaling. Described herein is a basic protocol for arrestin expression in Escherichia 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, 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 the overall salt concentration is maintained at or above physiological levels. © 2023 Wiley Periodicals LLC.Basic Protocol: Large‐scale expression and purification of arrestinsAlternate Protocol: Purification of arrestin‐3 and truncated form of arrestin‐1‐(1‐378)Support Protocol: Small‐scale test expression of wild‐type and mutant arrestins in E. coli