Rapid matrix-assisted refolding of histidine-tagged proteins...Solubilization Analytical techniques...

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


Workflow

Purification

Refolding

Solubilization

Analytical techniques for monitoring refolding

Protein refolding

Cell disruption

Freezing/storage

Cell harvest

Inclusion body preparation

Sedimentation and wash


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


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


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


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


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


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


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


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


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


Screening strategy

His MultiTrap™ FF

NaCl conc.

Additives

pH/buffersubstance

Stepwise optimization


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


Parallel matrix-assisted refolding

Equilibrate(Solubilization solution)

AnalyzeElute(Imidazole)

Incubate(1 hour, 20 ˚C)

Applyrefolding solution

Loadsolubilized protein

Wash(Solubilization solution)

Prepare plate


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


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


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


0

10

20

30

ab

cd

ef

gh

% re

fold

ing


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


Comparison - Time

Time (min) 0 20 40 60 120

Rel. refolding yield (%)

0

20

40

60

80

100

Dilution refolding


Citric synthase


Protein concentration (mg/ml)


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

(%)


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


Dilution refolding

Refo

ldin

g yi

eld

(%)


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


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)


Scale-upHis MultiTrap™ FF

HisTrap™ FF 1 ml

eGFP CS FNR GLK β-gal

Refolding yield (%)

0

20

40

60

80

100



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


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


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


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

Rapid matrix-assisted refolding of histidine-tagged proteins...Solubilization Analytical techniques...

Documents

Transcript of Rapid matrix-assisted refolding of histidine-tagged proteins...Solubilization Analytical techniques...