Rapid matrix-assisted refolding of histidine-tagged proteins

Outline Introduction Screening of refolding conditions Scale-up 2

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

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

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

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

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

General success data Refold web site: http://refold.med.monash.edu.au/analysis.php Refolding entries: 1157 Proteins: 758 (Sept 2009) Refolding techniques: Molecular weight: Dilution/dialysis refolding (83%) (~14%) 10-50 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%) 0-10 10-20 20-30 30-40 Number of entries 40-50 50-60 60-70 70-80 80-90 90-100 >100 Trimer (1.6 %) 5 to 14-mer (0.9 %) ph Dimer (15 %) Unkown (35.7 %) Monomer 44% (44.7 %) 5.0-5.5 5.5-6.0 6.0-6.5 6.5-7.0 7.0-7.5 7.5-8.0 8.0-8.5 8.5-9.0 9.0-9.5 9.5-10.0 10.0-10.5 10.5-11.0 8/ Aug 2009

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

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

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

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, 869-876 12/ Aug 2009

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

Screening strategy Stepwise optimization ph/buffer substance NaCl conc. Additives His MultiTrap FF 14/ Aug 2009

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

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

Proteins 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 17/ Aug 2009

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 8.0 200-300 mm NaCl Mixture of 40-50 mm of each Arg and Gln Reducing agents (DTT, TCEP) 18/ Aug 2009

Additives in best of ph and NaCl % refolding 100 80 60 40 20 0 Citrate synthase 0 50 6070 40 30 20 10 Arg + Gln [mm] % refolding 100 80 60 40 20 0 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

Refolding of a tetrameric protein ß-galactosidase 464 kda (4 x 116 kda) 30 40 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

Comparison - Time Rel. refolding yield (%) 100 80 Dilution refolding Citric synthase 60 40 20 0 0 20 40 60 120 Time (min) 21/ Aug 2009

Comparison - Protein concentration Citric synthase Refolding yield (%) 100 80 60 40 20 0 0 2 4 6 8 10 12 14 16 Protein concentration (mg/ml) Refolding yield (%) Dilution refolding 40 30 20 10 0 0 5 10 15 20 25 30 35 Protein concentation (µg/ml) 22/ Aug 2009

100 Dilution refolding Refolding yield (%) 80 60 40 20 % 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

On-column refolding mau 1000 900 load Load refolding Refolding Elution elution Glucokinase 800 700 600 pause Pause 500 400 refolding refolding elution F2 X1 X2 X3 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 0 5 10 15 20 25.0 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

Scale-up His MultiTrap FF HisTrap FF 1 ml Refolding yield (%) 100 80 60 40 20 0 egfp CS FNR GLK β-gal 26/ Aug 2009

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

Acknowledgements Johannes Buchner Martin Haslbeck Tetanya Dashivets Munich folding factory Technische Universität München, Germany 28/ Aug 2009

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. 29 / 2008

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