Purification of capping protein using the capping protein binding site of CARMIL as an affinity matrix.
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.
Remmert
Kirsten
K
Laboratory of Cell Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Uruno
Takehito
T
Hammer
John A
JA
D000048:Acanthamoeba / Q000737:chemistry; D051344:Actin Capping Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002457:Cell Extracts / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D051379:Mice; D008840:Microfilament Proteins; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry
D051344:Actin Capping Proteins; C504800:Carmil1 protein, mouse; D002352:Carrier Proteins; D002457:Cell Extracts; D008840:Microfilament Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D012685:Sepharose