Purification of capping protein using the capping protein binding site of CARMIL as an affinity matrix

Kirsten Remmert; Takehito Uruno; John A. Hammer

doi:10.1016/j.pep.2009.05.002

Purification of capping protein using the capping protein binding site of CARMIL as an affinity matrix

Kirsten Remmert, Takehito Uruno, John A. Hammer

Research output: Contribution to journal › Article › peer-review

4 Citations (Scopus)

Abstract

Capping protein (CP) is a ubiquitously expressed, heterodimeric actin binding protein that is essential for normal actin dynamics in cells. The existing methods for purifying native CP from tissues and recombinant CP from bacteria are time-consuming processes that involve numerous conventional chromatographic steps and functional assays to achieve a homogeneous preparation of the protein. Here, we report the rapid purification of Acanthamoeba CP from amoeba extracts and recombinant mouse CP from E. coli extracts using as an affinity matrix GST-fusion proteins containing the CP binding site from Acanthamoeba CARMIL and mouse CARMIL-1, respectively. This improved method for CP purification should facilitate the in vitro analysis of CP structure, function, and regulation.

Original language	English
Pages (from-to)	113-119
Number of pages	7
Journal	Protein Expression and Purification
Volume	67
Issue number	2
DOIs	https://doi.org/10.1016/j.pep.2009.05.002
Publication status	Published - Oct 2009
Externally published	Yes

All Science Journal Classification (ASJC) codes

Biotechnology

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10.1016/j.pep.2009.05.002

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abstract = "Capping protein (CP) is a ubiquitously expressed, heterodimeric actin binding protein that is essential for normal actin dynamics in cells. The existing methods for purifying native CP from tissues and recombinant CP from bacteria are time-consuming processes that involve numerous conventional chromatographic steps and functional assays to achieve a homogeneous preparation of the protein. Here, we report the rapid purification of Acanthamoeba CP from amoeba extracts and recombinant mouse CP from E. coli extracts using as an affinity matrix GST-fusion proteins containing the CP binding site from Acanthamoeba CARMIL and mouse CARMIL-1, respectively. This improved method for CP purification should facilitate the in vitro analysis of CP structure, function, and regulation.",

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AU - Uruno, Takehito

AU - Hammer, John A.

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AB - Capping protein (CP) is a ubiquitously expressed, heterodimeric actin binding protein that is essential for normal actin dynamics in cells. The existing methods for purifying native CP from tissues and recombinant CP from bacteria are time-consuming processes that involve numerous conventional chromatographic steps and functional assays to achieve a homogeneous preparation of the protein. Here, we report the rapid purification of Acanthamoeba CP from amoeba extracts and recombinant mouse CP from E. coli extracts using as an affinity matrix GST-fusion proteins containing the CP binding site from Acanthamoeba CARMIL and mouse CARMIL-1, respectively. This improved method for CP purification should facilitate the in vitro analysis of CP structure, function, and regulation.

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Purification of capping protein using the capping protein binding site of CARMIL as an affinity matrix

Abstract

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