Rapid large-scale purification of myofilament proteins using a cleavable His6-tag Journal Article

Local Library Link: Find It @ Loyola

Authors: Zhang, M; Martin, J. L.; Kumar, M; Khairallah, R. J.; de Tombe, P. P.
Article Title: Rapid large-scale purification of myofilament proteins using a cleavable His6-tag
Abstract: With the advent of high-throughput DNA sequencing, the number of identified cardiomyopathy-causing mutations has increased tremendously. As the majority of these mutations affect myofilament proteins, there is a need to understand their functional consequence on contraction. Permeabilized myofilament preparations coupled with protein exchange protocols are a common method for examining into contractile mechanics. However, producing large quantities of myofilament proteins can be time consuming and requires different approaches for each protein of interest. In the present study, we describe a unified automated method to produce troponin C, troponin T, and troponin I as well as myosin light chain 2 fused to a His6-tag followed by a tobacco etch virus (TEV) protease site. TEV protease has the advantage of a relaxed P1' cleavage site specificity, allowing for no residues left after proteolysis and preservation of the native sequence of the protein of interest. After expression in Esherichia coli, cells were lysed by sonication in imidazole-containing buffer. The His6-tagged protein was then purified using a HisTrap nickel metal affinity column, and the His6-tag was removed by His6-TEV protease digestion for 4 h at 30 degrees C. The protease was then removed using a HisTrap column, and complex assembly was performed via column-assisted sequential desalting. This mostly automated method allows for the purification of protein in 1 day and can be adapted to most soluble proteins. It has the advantage of greatly increasing yield while reducing the time and cost of purification. Therefore, production and purification of mutant proteins can be accelerated and functional data collected in a faster, less expensive manner.
Journal Title: American journal of physiology.Heart and circulatory physiology
Volume: 309
Issue: 9
ISSN: 1522-1539; 0363-6135
Publisher: the American Physiological Society  
Journal Place: United States
Date Published: 2015
Start Page: H1509
End Page: 15
Language: eng
DOI/URL:

10.1152/ajpheart.00598.2015
Notes: CI: Copyright (c) 2015; JID: 100901228; OTO: NOTNLM; 2015/07/28 [received]; 2015/09/15 [accepted]; 2015/09/18 [aheadofprint]; ppublish