Expression and purification of full-length mouse CARM1 from transiently transfected HEK293T cells using HaloTag technology.

Abstract

Coactivator-associated arginine methyl transferase 1 (CARM1) is a protein arginine methyltransferase (PRMT) family member that functions as a coactivator in androgen and estrogen signaling pathways and plays a role in the progression of prostate and breast cancer. CARM1 catalyzes methylation of diverse protein substrates. Prior attempts to purify the full-length mouse CARM1 protein have proven unsatisfactory. The full-length protein expressed in Escherichia coli forms insoluble inclusion bodies that are difficult to denature and refold. The presented results demonstrate the use of a novel HaloTag™ technology to purify full-length CARM1 from both E. coli and mammalian HEK293T cells. A small amount of CARM1 was purified from E. coli; however, the protein was truncated on the N-terminus by 10-50 amino acids, most likely due to endogenous proteolytic activity. In contrast, substantial quantities of soluble full-length CARM1 were purified from transiently transfected HEK293T cells. The CARM1 from HEK293T cells was isolated alongside a number of co-purifying interacting proteins. The covalent bond formed between the HaloTag and the HaloLink resin allowed the use of stringent wash conditions without risk of eluting the CARM1 protein. The results also illustrate a highly effective approach for purifying and enriching both CARM1-associated proteins as well as substrates for CARM1's methyltransferase activity.

• Authors: Robert S Chumanov, Peter A Kuhn, Wei Xu, Richard R Burgess
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 76 Issue 2 Pg. 145-53 (Apr 2011) ISSN: 1096-0279 [Electronic] United States
• PMID: 21126607 (Publication Type: Comparative Study, Journal Article, Research Support, N.I.H., Extramural)
• Copyright: Copyright © 2010 Elsevier Inc. All rights reserved.

Chemical References

• Immobilized Proteins
• Recombinant Fusion Proteins
• Protein-Arginine N-Methyltransferases
• coactivator-associated arginine methyltransferase 1

Topics

• Animals
• Blotting, Western
• Chromatography, Affinity (methods)
• Cloning, Molecular
• Electrophoresis, Polyacrylamide Gel
• Escherichia coli (genetics, metabolism)
• HEK293 Cells
• Humans
• Immobilized Proteins (metabolism)
• Mice
• Microscopy, Fluorescence
• Protein-Arginine N-Methyltransferases (biosynthesis, genetics, isolation & purification)
• Recombinant Fusion Proteins (biosynthesis, genetics, isolation & purification)
• Solubility
• Transfection