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

Results Methods

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

rPKA fragment

Phospho-rTnI

rPKA fragment

rPKA fragment

Total rTnI

PKA Concentration (µl/ml)

0 1 5 10 50 100

Recombinant His6-PKA

Commercial PKA (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

Ab

so

rban

ce

(mA

U)

Co

nd

ucti

vit

y(m

S/ cm

)

Cumulative Volume (ml)

0

20

40

60

80

100

Co

ncen

trati

on

(%E

luent)

Tn Complex

TnCTnI

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 runLane 3: After Protease DigestLane 4: After 2nd FPLC run

Purification of Myofilament Proteins His-rPKA can phosphorylate TnI

TEV Protease • Native Cleavage Sequence is

ENLYFQ\G

• Will cleave at EXLYΦQ\φ

– X is any residue

– Φ Large hydrophobic

– φ small hydrophobic

P1’ specificity of TEV protease

Transform and grow bacteria

Lysis bacterial pellet6M Urea

Load on HisTrapcolumn and elute

His-tagged protein

Perform buffer exchange with

desalting columnDigestion buffer

Mix TnT, TnI and TnC in equal

amounts

Fold proteins into complex by

sequential dialysisRelax buffer

Digest His-tag with TEV Protease

Load on MonoQcolumn collect peak fractions eluting past

0.4 M KCl

Load on HisTrap and collect flowthrough

Perform buffer exchange on f lowthrough

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