rapid matrix-assisted refolding of histidine-tagged proteins...solubilization analytical techniques...
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Rapid matrix-assisted refolding of histidine-tagged proteins
2
Outline
Introduction
Screening of refolding conditions
Scale-up
3/ Aug 2009Matrix-assisted refolding
Inclusion bodies
AdvantagesVery high expression levels
Relatively high purity already in the inclusion bodies
Protection from proteolytic enzymes
Simple preparation
Visible with phase-contrast microscope
Expression of toxic proteins
Electron micrograph of E. coliwith inclusion bodiesBy courtesy of Prof. Jonathan King, MIT, Cambridge
Light-microscopic image of E. coli with inclusion bodies
4/ Aug 2009Matrix-assisted refolding
Workflow
Purification
Refolding
Solubilization
Analytical techniques for monitoring refolding
Protein refolding
Cell disruption
Freezing/storage
Cell harvest
Inclusion body preparation
Sedimentation and wash
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Principles
Aggregation competes with folding
Denaturantconcentration
Inclusionbodies
Unfolded (U)
Native (N)
Foldingintermediates (I)
Amount ofstructure
Aggregates (A)
Compact and rigid Open and flexible
Low High
Low
High
Nativeprotein
Foldingintermediate
Unfoldedpolypeptide
Aggregate
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Additives in refolding buffersEffects
Additives Protein structure Intra- and inter-molecular interactions
Denaturants Urea Guanidine-HClStrong detergent
Destabilized Disrupted
Urea (low conc.) Guanidine-HCl (low conc.) Arginine-HCl
Neutral Reduced
Reducing agents DTT, DTE, TCEP, GSH Reduced S-S bridges
Aggregation suppressors
Mild detergentsPEGsProlineCyclodextrins
Neutral Reduced
Folding enhancers
SugarsPolyolsAmmonium sulfateMagnesium chlorideGlycineAlanine
Stabilized Enhanced
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Analysis
Protein properties Techniques
Protein function Enzyme activity assayBinding activity
Size and molecular weight Gel filtrationGel filtration with MALLSNative PAGE
S-S-bridges Reversed phase chromatography (RPC)
Tertiary structure Intrinsic fluorescenceNMR
Secondary structure Circular dichroismChromatographic behavior (e.g., HIC, RPC or IEC)
Compactness of native state Limited proteolysis combined with SDS-PAGE
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Refolding entries: 1157Proteins: 758(Sept 2009)
General success data
Refolding techniques:
Dilution/dialysis refolding (83%)Matrix-assisted refolding (~14%)
Molecular weight: 10-50 kDa
Fusion proteins: Untagged proteins (60%)Histidine tagged proteins (24%)
No of disulfides: Mostly 0-3, >10 has been done
Oligomeric state: From monomer to 14-mer
Mr (x10-3)
70-80
10-20
20-30
30-40
0-10
60-70
50-60
40-50
80-90
90-100
>100
Number of entries
Number of entries
pH
5.0-5.5
5.5-6.0
6.0-6.5
6.5-7.0
7.0-7.5
7.5-8.0
10.0-10.5
8.0-8.5
8.5-9.0
9.5-10.0
9.0-9.5
10.5-11.0
44%
Unkown (35.7 %)
Monomer (44.7 %)Dimer (15 %)
Trimer (1.6 %)
Tetramer (2.1%)
5 to 14-mer (0.9 %)
Refold web site: http://refold.med.monash.edu.au/analysis.php
9/ Aug 2009Matrix-assisted refolding
Refolding techniques
Technique Pros (+) Cons (-)Dilution Simple
InexpensiveSlow refoldingLow protein concentrationLarge final volumes
Dialysis SimpleInexpensive
Slow refoldingLow protein concentrationLarge volumes of buffers
Matrix-assisted refolding
Fast refoldingHigh protein concentrationImmobilization reduces aggregationOne step purification and refoldingLimited buffer consumption
May require affinity tagCompatibility with solubilizationconditions required
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Matrix-assisted protein refoldingTechniques
Immobilized metal ion affinity chromatography (IMAC)
Gel filtration (GF)
Ion-exchange chromatography (IEC)
Hydrophobic interaction chromatography (HIC)
Immobilized catalysts and artificial chaperones
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Matrix-assisted refolding and purification using an IMAC column
Elution gradient:20 mM to 500 mM imidazole
Refolding gradient:6 M to 0 M urea
Volume
A280
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Development of methods for matrix-assisted protein refolding
Munichfoldingfactory
Technische Universität München, Germany
Ref: Rapid matrix-assisted refolding of histidine tagged proteins Dashivets et. al., ChemBioChem 2009, 10, 869-876
Johannes BuchnerMartin HaslbeckTetanya Dashivets
13/ Aug 2009Matrix-assisted refolding
Workflow Inclusion body preparation
Purification
Refolding
Solubilization
Analytical technique for monitoring refolding
Inclusion bodies
Supernatant with soluble target protein
Screening
Purification
Refolding
Solubilization
Scale-up
Purification 1 2
Reference sample
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Screening strategy
His MultiTrap™ FF
NaCl conc.
Additives
pH/buffersubstance
Stepwise optimization
15/ Aug 2009Matrix-assisted refolding
Areas of use: Small-scale protein purificationCondition screening and optimizationExpression screening
Amount resin/well: 50 µl Ni Sepharose™ Fast Flow
Capacity/well: 800 µg histidine-tagged proteins
Well volume: 800 µl
Handling: CentrifugationVacuumManually or automatic
His MultiTrap™ FF
16/ Aug 2009Matrix-assisted refolding
Parallel matrix-assisted refolding
Equilibrate(Solubilization solution)
AnalyzeElute(Imidazole)
Incubate(1 hour, 20 ˚C)
Applyrefolding solution
Loadsolubilized protein
Wash(Solubilization solution)
Prepare plate
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Protein kDa pI Analysis Structure
Enhanced Green Fluorescent Protein (eGFP)
28 5.7 Fluorescence emission
Monomer
Ferredoxin-NADP+ reductase(FNR)
35 6.2 Enzyme assay Monomer
Glucokinase (GLK) 72 6.1 Enzyme assay Dimer
Citrate synthase (CS) 98 8.1 Enzyme assay Dimer
Beta-galactosidase (ß-Gal) 464 5.3 Enzyme assay Tetramer
Proteins
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Step-wise selection of conditions
Best of refolding buffer conditionsTris and phosphate buffers at pH 7.5 and 8.0200-300 mM NaClMixture of 40-50 mM of each Arg and GlnReducing agents (DTT, TCEP)
Buffer + pH NaCl Arg + Gln Additives
Ferredoxin NADP+ reductase
Buffers
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Additives in “best of pH and NaCl”
Additives a. 100 mM Sucroseb. 200 mM Sucrosec. 1% PEG 6000 d. 5% Glycerol e. 5 mM Cyclodextrinf. 10 mM Cyclodextring. 2 mM DTEh. 2 mM TCEPi. 5 mM TCEP
0
20
40
60
80
100
ab
cd
ef
ghi
% re
fold
ing
0
20
40
60
80
100
010
2030
4050
6070
% re
fold
ing
Arg
+ Gln
[mM
]
Glucokinase
Additives0 - 70 mM (Arginine-HCl + Glutamine)
Citrate synthase
20/ Aug 2009Matrix-assisted refolding
0
10
20
30
ab
cd
ef
gh
% re
fold
ing
Matrix-assisted refolding
Dilution refolding
ß-galactosidase464 kDa (4 x 116 kDa)
Refolding of a tetrameric protein
40 mM Na-Phos200 mM NaCl40 mM Arg/GlnpH 7.5
a. 2 mM DTEb. 5 mM DTEc. 2 mM TCEPd. 5 mM TCEP
100 mM Tris/HCl300 mM NaCl40 mM Arg/ GlnpH 7.5
e. 2 mM DTEf. 5 mM DTEg. 2 mM TCEPh. 5 mM TCEP
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Comparison - Time
Time (min) 0 20 40 60 120
Rel. refolding yield (%)
0
20
40
60
80
100
Dilution refolding
Matrix-assisted refolding
Citric synthase
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Protein concentration (mg/ml)
Matrix-assisted refolding
0 2 4 6 8 10 12 14 160
20
40
60
80
100
0 5 10 15 20 25 30 350
10
20
30
40
Comparison - Protein concentrationCitric synthase
Protein concentation (µg/ml)
Dilution refolding
Refo
ldin
g yi
eld
(%)
Refo
ldin
g yi
eld
(%)
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Monomer 28 kDa
Homodimer98 kDa
(2x49 kDa)
Homodimer72 kDa
(2x36 kDa)
Homotetramer464 kD
(4x116 kDa)
Monomer35 kDa
eGFP CS FNR GLK ß-Gal
% re
fold
ing
0
20
40
60
80
100
Matrix-assisted refolding
Dilution refolding
Refo
ldin
g yi
eld
(%)
24/ Aug 2009Matrix-assisted refolding
Workflow Inclusion body preparation
Purification
Refolding
Solubilization
Analytical technique for monitoring refolding
Inclusion bodies
Supernatant with soluble target protein
Screening
Purification
Refolding
Solubilization
Scale-up
Purification 1 2
Reference sample
25/ Aug 2009Matrix-assisted refolding
On-column refolding
System: ÄKTApurifier™Column: HisTrap™ FF 1 mlFlow rate: 0.5 ml/minDenaturing buffer: 40 mM Na-phosphate, 300 mM NaCl, 6 M Gu-HCl, pH 8.0Equilibration buffer: 40 mM Na-phosphate, 300 mM NaCl, 5 mM imidazole, pH 8.0Refolding buffer: 40 mM Na-phosphate, 200 mM NaCl, 50 mM Arg-HCl, 50 mM Gln, 5 mM TCEP, pH 8.0Refolding pause: 1 hourGradient elution: 5 to 500 mM imidazole in equilibration buffer
Glucokinase
load refolding elution
paus
e
15 25.0 mlre
fold
ing
refo
ldin
g
elut
ion
F2 X1 X2 X3 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12
Load Refolding Elution
Pause
400
500
600
700
800
900
1000
0 5 10 15 20 25.0 mlre
fold
ing
refo
ldin
g
elut
ion
F2 X1 X2 X3 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12
mAU
Volume (ml)
26/ Aug 2009Matrix-assisted refolding
Scale-upHis MultiTrap™ FF
HisTrap™ FF 1 ml
eGFP CS FNR GLK β-gal
Refolding yield (%)
0
20
40
60
80
100
27/ Aug 2009Matrix-assisted refolding
Matrix-assisted refolding
Fast
High protein concentrations
High yield even for a tetrameric protein
Screening of refolding conditions in parallel using His MultiTrap™ FF 96-well filter plates
Scale-up of best conditions using pre-packed HisTrap™ FF column
Summary
28/ Aug 2009Matrix-assisted refolding
Johannes BuchnerMartin HaslbeckTetanya Dashivets
Munich folding factoryTechnische UniversitätMünchen, Germany
Acknowledgements
29 / 2008
MultiTrap, HisTrap, Sepharose and ÄKTApurifier are trademarks of GE Healthcare companies. GE, imagination at work and GE monogram are trademarks of General Electric Company.
Purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues may require a license under US pat 5,284,933 and US pat 5,310,663 , including corresponding foreign patents (assigne: Hoffman La Roche, Inc).
All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information.
© 2009 General Electric Company – All rights reserved.First published September 2009.
GE Healthcare Bio-Sciences AB, a General Electric Company.
GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden.
30 / 2008
Matrix-assisted refolding
Fast
High protein concentrations
High yield even for a tetrameric protein
Screening of refolding conditions in parallel using His MultiTrap™ FF 96-well filter plates
Scale-up of best conditions using pre-packed HisTrap™ FF column
Summary
31/ Aug 2009Matrix-assisted refolding
Tips
Prepare refolding buffers fresh prior to performing assayAdjust pH of buffers after the addition of all components. (L-Arginine-HCl
increases pH dramatically)Weigh in additives (If unstable avoid stock solution)Do not add reducing agents prior to pH adjustment. (pH electrode may
accelerate oxidation)Triplicate samplesPerform buffer exchange if “additives in buffer” disturb the enzyme assayIf the protein is unstable – perform the assay immediately after elution