1. RAPID, HIGH EFFICIENCY PURIFICATION OF MYOFILAMENT PROTEINS USING
TOBACCO ETCH VIRUS PROTEASE
Mengjie Zhang, Jody L. Martin, Pieter P. De Tombe, Ramzi J. Khairallah
Loyola University Chicago, Dept. of Cell and Molecular Physiology, Maywood, IL, USA.
Abstract
Introduction
ResultsMethods
As more and more cardiomyopathy causing mutations are
identified, functional studies investigating contractility and
myofilament mechanics with mutated proteins become
more crucial. Several methods are available, but most rely
on exchanging recombinant protein into permeabilized
heart cells or myofilament preparation. However, success
requires large amounts of highly purified myofilament
proteins, a process which is usually different for each
individual protein. Here, we describe one single method
that can be used for purifying myofilament protein,
including troponin T, I and C, as well as myosin light chain
2, which has also been adapted to other recombinant
proteins.
• Previous traditional purification methods
• TnC: Cellulose DEAE (D52) anion exchange followed
by a Phenyl Sepharose hydrophobic interaction
• TnI: CM Sepharose weak cation followed by a custom
TnC capture column
• TnT: Cellulose DEAE (DEAE FF) anion exchange
• Disadvantages
• Time-consuming: 3 days purification + 3 days dialysis
• Labor intensive: 10 different buffers for the three
troponins
• Low yield: 1-3 mg target protein per liter of bacterial
culture.
• Kobayashi T, Solaro RJ. Increased Ca2+ affinity of cardiac thin
filaments reconstituted with cardiomyopathy-related mutant cardiac
troponin I. J Biol Chem. 2006
• Biesiadecki BJ, Kobayashi T, Walker JS, Solaro RJ, de Tombe PP.
The troponin C G159D mutation blunts myofilament desensitization
induced by troponin I Ser23/24 phosphorylation. Circ Res. 2007.
• Tropea JE, Cherry S, Waugh DS. Expression and purification of
soluble His6-tagged TEV protease. Methods in Mol Biol. 2009.
• Golovanov AP, Hautbergue GM, Wilson SA, Lian LY. A simple
method for improving protein solubility and long-term stability. J Am
Chem Soc. 2004.
• Morjana N, Tal R. Expression and equilibrium denaturation of
cardiac troponin I: stabilization of a folding intermediate during
denaturation by urea. Biotechnol Appl Biochem. 1998;28:7-17.
References & Acknowledgements
General workflow for the purification of
His-tagged proteins.
Schematic of pET 28 vector for expression
of target proteins.
ÄKTA FPLC Automated Protein Purification
System
PKA Concentration (µl/ml)
0 1 5 10 50 100 500 1000
0 ATP
5mM ATP
Total Protein
rPKAfragment
Phospho-rTnI
rPKAfragment
rPKAfragment
Total rTnI
PKA Concentration(µl/ml)
0 1 5 10 50 100
RecombinantHis6-PKA
CommercialPKA
(from bovine heart)
• Goal: Employ a cleavable tag (His6) to help with the
purification without altering the native sequence of the
protein
•Improvements
• Much more rapid: ~2 days purification
•Lower labor cost (automated, 4 buffers only)
• Increased yield: TnC: ~30 mg per liter culture;
TnT and TnI: ~15 mg per liter culture.
Unified purification procedure
1st FPLC run – His-Tag purification of TnT 2nd FPLC run – Removal of protease
0 20 40 60
0
10
20
0
50
100
150
Absorbance
(mAU)
Conductivity
(mS/cm)
Cumulative Volume (ml)
0
20
40
60
80
100
Concentration
(%Eluent)
Tn Complex
TnC
TnI
Elution with KCl
3rd FPLC run – Complex Purification
75
50
37
25
20
15
10
cTnT-myc
cTnI
cTnC
Troponin complex
Eluted Fractions
1 2 3 4 5 6 7 8 9 10 11 12
Troponin C Troponin TTroponin I
PKAαMLC2
50
37
25
20
15
10
50
37
25
20
15
10
50
37
25
20
15
10
M 1 2 3 4 M 1 2 3 4 M 1 2 3 4
50
37
25
20
15
10
M 1 2
50
37
25
20
15
10
M 1 2 3 4
Lane 1:Bacterial Lysate
Lane 2:After 1st
FPLC run
Lane 3:After Protease Digest
Lane 4:After 2nd FPLC run
Purification of Myofilament Proteins His-rPKA can phosphorylate TnI
TEV Protease
• Native Cleavage Sequence is
ENLYFQG
• Will cleave at EXLYΦQφ
– X is any residue
– Φ Large hydrophobic
– φ small hydrophobic
P1’ specificity of TEV protease
Transform and grow
bacteria
Lysis bacterial pellet
6M Urea
Load on HisTrap
column and elute
His-tagged protein
Perform buffer
exchange with
desalting column
Digestion buffer
Mix TnT, TnI and
TnC in equal
amounts
Fold proteins into
complex by
sequential dialysis
Relax buffer
Digest His-tag with
TEV Protease
Load on MonoQ
column collect peak
fractions eluting past
0.4 M KCl
Load on HisTrap and
collect flowthrough
Perform buffer
exchange on
flowthrough
6M Urea
Store proteins in 6M
Urea at -80ºC for
further use
Concentrate using
spin column to
2mg/ml
1st FPLC run
2nd FPLC run
3rd FPLC run