Expression of Untagged Arrestins in E. coli and Their Purification.
arrestin
chromatography
expression
purification
recombinant
Journal
Current protocols
ISSN: 2691-1299
Titre abrégé: Curr Protoc
Pays: United States
ID NLM: 101773894
Informations de publication
Date de publication:
Sep 2023
Sep 2023
Historique:
pmc-release:
01
09
2024
medline:
7
9
2023
pubmed:
6
9
2023
entrez:
6
9
2023
Statut:
ppublish
Résumé
Purified 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 arrestins Alternate 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.
Identifiants
pubmed: 37671938
doi: 10.1002/cpz1.832
pmc: PMC10491425
mid: NIHMS1919181
doi:
Substances chimiques
Arrestin
0
Arrestins
0
Ammonium Sulfate
SU46BAM238
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e832Subventions
Organisme : NEI NIH HHS
ID : R01 EY011500
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM122491
Pays : United States
Informations de copyright
© 2023 Wiley Periodicals LLC.
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