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°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.
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University, Chicago, Illinois.
authors.forename: Mengjie
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University, Chicago, Illinois.
authors.forename: Jody L
authors.initials: JL
authors.lastname: Martin
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University, Chicago, Illinois.
authors.forename: Mohit
authors.initials: M
authors.lastname: Kumar
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University, Chicago, Illinois ramzi.khairallah@live.com.
authors.forename: Ramzi J
authors.initials: RJ
authors.lastname: Khairallah
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University, Chicago, Illinois.
authors.forename: Pieter P
authors.initials: PP
authors.lastname: de Tombe
mesh_terms: D001331:Automation; D024722:Cardiac Myosins / Q000096:biosynthesis* / Q000235:genetics; D002846:Chromatography, Affinity; D003911:Dextrans; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine; D006801:Humans; D009210:Myofibrils; D018994:Myosin Light Chains / Q000096:biosynthesis* / Q000235:genetics; D009532:Nickel; D009842:Oligopeptides; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012685:Sepharose; D019209:Troponin C / Q000096:biosynthesis* / Q000235:genetics; D019210:Troponin I / Q000096:biosynthesis* / Q000235:genetics; D020107:Troponin T / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D003911:Dextrans; C471213:His-His-His-His-His-His; D018994:Myosin Light Chains; D009842:Oligopeptides; D011994:Recombinant Proteins; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; C107184:myosin light chain 2; D006639:Histidine; D009532:Nickel; D012685:Sepharose; C025614:sephadex; D010450:Endopeptidases; C099488:TEV protease; D024722:Cardiac Myosins
keywords: His-tag; protein purification; tobacco etch virus protease; troponin