Rapid matrix-assisted refolding of histidine-tagged proteins
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1 Rapid matrix-assisted refolding of histidine-tagged proteins
2 Outline Introduction Screening of refolding conditions Scale-up 2
3 Inclusion bodies Advantages Very 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. coli with inclusion bodies By courtesy of Prof. Jonathan King, MIT, Cambridge Light-microscopic image of E. coli with inclusion bodies 3/ Aug 2009
4 Workflow Inclusion body preparation Cell harvest Protein refolding Solubilization Cell disruption Purification Sedimentation and wash Refolding Freezing/storage Analytical techniques for monitoring refolding 4/ Aug 2009
5 Principles Amount of structure High Compact and rigid Native (N) Open and flexible Folding intermediates (I) Aggregates (A) Inclusion bodies Unfolded (U) Low Low Denaturant concentration High Unfolded polypeptide Folding intermediate Native protein Aggregate Aggregation competes with folding 5/ Aug 2009
6 Additives in refolding buffers Effects Additives Protein structure Intra- and intermolecular interactions Denaturants Urea Guanidine-HCl Strong detergent Destabilized Disrupted Aggregation suppressors Urea (low conc.) Guanidine-HCl (low conc.) Arginine-HCl Neutral Reduced Mild detergents PEGs Proline Cyclodextrins Neutral Reduced Folding enhancers Sugars Polyols Ammonium sulfate Magnesium chloride Glycine Alanine Stabilized Enhanced Reducing agents DTT, DTE, TCEP, GSH Reduced S-S bridges 6/ Aug 2009
7 Analysis Protein properties Protein function Size and molecular weight S-S-bridges Tertiary structure Secondary structure Compactness of native state Enzyme activity assay Binding activity Gel filtration Gel filtration with MALLS Native PAGE Techniques Reversed phase chromatography (RPC) Intrinsic fluorescence NMR Circular dichroism Chromatographic behavior (e.g., HIC, RPC or IEC) Limited proteolysis combined with SDS-PAGE 7/ Aug 2009
8 General success data Refold web site: Refolding entries: 1157 Proteins: 758 (Sept 2009) Refolding techniques: Molecular weight: Dilution/dialysis refolding (83%) (~14%) kda Number of entries M r (x10-3 ) Fusion proteins: Untagged proteins (60%) Histidine tagged proteins (24%) No of disulfides: Oligomeric state: Mostly 0-3, >10 has been done From monomer to 14-mer Tetramer (2.1%) Number of entries >100 Trimer (1.6 %) 5 to 14-mer (0.9 %) ph Dimer (15 %) Unkown (35.7 %) Monomer 44% (44.7 %) / Aug 2009
9 Refolding techniques Technique Pros (+) Cons (-) Dilution Dialysis Matrixassisted refolding Simple Inexpensive Simple Inexpensive Fast refolding High protein concentration Immobilization reduces aggregation One step purification and refolding Limited buffer consumption Slow refolding Low protein concentration Large final volumes Slow refolding Low protein concentration Large volumes of buffers May require affinity tag Compatibility with solubilization conditions required 9/ Aug 2009
10 Matrix-assisted protein refolding Techniques Immobilized metal ion affinity chromatography (IMAC) Gel filtration (GF) Ion-exchange chromatography (IEC) Hydrophobic interaction chromatography (HIC) Immobilized catalysts and artificial chaperones 10/ Aug 2009
11 and purification using an IMAC column Refolding gradient: 6 M to 0 M urea Elution gradient: 20 mm to 500 mm imidazole A 280 Volume 11/ Aug 2009
12 Development of methods for matrixassisted protein refolding Johannes Buchner Martin Haslbeck Tetanya Dashivets Munich folding factory Technische Universität München, Germany Ref: Rapid matrix-assisted refolding of histidine tagged proteins Dashivets et. al., ChemBioChem 2009, 10, / Aug 2009
13 Workflow Inclusion body preparation Supernatant with soluble target protein Inclusion bodies Purification 1 2 Screening Scale-up Solubilization Solubilization Reference sample Purification Refolding Purification Refolding Analytical technique for monitoring refolding 13/ Aug 2009
14 Screening strategy Stepwise optimization ph/buffer substance NaCl conc. Additives His MultiTrap FF 14/ Aug 2009
15 His MultiTrap FF Areas of use: Amount resin/well: Capacity/well: Small-scale protein purification Condition screening and optimization Expression screening 50 µl Ni Sepharose Fast Flow 800 µg histidine-tagged proteins Well volume: 800 µl Handling: Centrifugation Vacuum Manually or automatic 15/ Aug 2009
16 Parallel matrix-assisted refolding Prepare plate Equilibrate (Solubilization solution) Load solubilized protein Wash (Solubilization solution) Apply refolding solution Incubate (1 hour, 20 C) Elute (Imidazole) Analyze 16/ Aug 2009
17 Proteins Protein kda pi Analysis Structure Enhanced Green Fluorescent Protein (egfp) Fluorescence emission Monomer Ferredoxin-NADP + reductase (FNR) Enzyme assay Monomer Glucokinase (GLK) Enzyme assay Dimer Citrate synthase (CS) Enzyme assay Dimer Beta-galactosidase (ß-Gal) Enzyme assay Tetramer 17/ Aug 2009
18 Step-wise selection of conditions Ferredoxin NADP + reductase Buffer + ph NaCl Arg + Gln Additives Buffers Best of refolding buffer conditions Tris and phosphate buffers at ph 7.5 and mm NaCl Mixture of mm of each Arg and Gln Reducing agents (DTT, TCEP) 18/ Aug 2009
19 Additives in best of ph and NaCl % refolding Citrate synthase Arg + Gln [mm] % refolding Glucokinase g hi d ef a bc Additives a. 100 mm Sucrose b. 200 mm Sucrose c. 1% PEG 6000 d. 5% Glycerol e. 5 mm Cyclodextrin f. 10 mm Cyclodextrin g. 2 mm DTE h. 2 mm TCEP i. 5 mm TCEP Additives 0-70 mm (Arginine-HCl + Glutamine) 19/ Aug 2009
20 Refolding of a tetrameric protein ß-galactosidase 464 kda (4 x 116 kda) mm Na-Phos 200 mm NaCl 40 mm Arg/Gln ph 7.5 a. 2 mm DTE b. 5 mm DTE c. 2 mm TCEP d. 5 mm TCEP 20 % refolding 10 0 a e f g h d c b 100 mm Tris/HCl 300 mm NaCl 40 mm Arg/ Gln ph 7.5 e. 2 mm DTE f. 5 mm DTE g. 2 mm TCEP h. 5 mm TCEP Dilution refolding 20/ Aug 2009
21 Comparison - Time Rel. refolding yield (%) Dilution refolding Citric synthase Time (min) 21/ Aug 2009
22 Comparison - Protein concentration Citric synthase Refolding yield (%) Protein concentration (mg/ml) Refolding yield (%) Dilution refolding Protein concentation (µg/ml) 22/ Aug 2009
23 100 Dilution refolding Refolding yield (%) % refolding Matrix-assisted refolding 0 egfp CS FNR GLK ß-Gal Monomer 28 kda Homodimer 98 kda (2x49 kda) Monomer 35 kda Homodimer 72 kda (2x36 kda) Homotetramer 464 kd (4x116 kda) 23/ Aug 2009
24 Workflow Inclusion body preparation Supernatant with soluble target protein Inclusion bodies Purification 1 2 Screening Scale-up Solubilization Solubilization Reference sample Purification Refolding Purification Refolding Analytical technique for monitoring refolding 24/ Aug 2009
25 On-column refolding mau load Load refolding Refolding Elution elution Glucokinase pause Pause refolding refolding elution F2 X1 X2 X3 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A ml Volume (ml) System: ÄKTApurifier Column: HisTrap FF 1 ml Flow rate: 0.5 ml/min Denaturing buffer: 40 mm Na-phosphate, 300 mm NaCl, 6 M Gu-HCl, ph 8.0 Equilibration buffer: 40 mm Na-phosphate, 300 mm NaCl, 5 mm imidazole, ph 8.0 Refolding buffer: 40 mm Na-phosphate, 200 mm NaCl, 50 mm Arg-HCl, 50 mm Gln, 5 mm TCEP, ph 8.0 Refolding pause: 1 hour Gradient elution: 5 to 500 mm imidazole in equilibration buffer 25/ Aug 2009
26 Scale-up His MultiTrap FF HisTrap FF 1 ml Refolding yield (%) egfp CS FNR GLK β-gal 26/ Aug 2009
27 Summary 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 27/ Aug 2009
28 Acknowledgements Johannes Buchner Martin Haslbeck Tetanya Dashivets Munich folding factory Technische Universität München, Germany 28/ Aug 2009
29 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 General Electric Company All rights reserved. First published September GE Healthcare Bio-Sciences AB, a General Electric Company. GE Healthcare Bio-Sciences AB, Björkgatan 30, SE Uppsala, Sweden. 29 / 2008
30 Summary 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 30 / 2008
31 Tips Prepare refolding buffers fresh prior to performing assay Adjust 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 samples Perform buffer exchange if additives in buffer disturb the enzyme assay If the protein is unstable perform the assay immediately after elution 31/ Aug 2009
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