china- high throughput screening for tagged...
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High throughput screening for tagged proteins and antibodies标签蛋白和抗体的高通量筛选
Gabriella Risberg Research engineerGE Healthcare, Sweden
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Outline
Introduction
Conditions screening
His-tagged proteins
Monoclonal antibodies
Refolding conditions for inclusion bodies
Summary
大纲
绪论
筛选条件
组氨酸标签蛋白
单克隆抗体
包涵体折叠条件
小结
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Many constructs → Many expression conditions → Many experiments
Challenging proteins 很多组成→很多表达条件→很多实验
– Standard protocols do not work 具有挑战性的蛋白
– Low expression levels --标准方案不起作用
– Membrane proteins --膜蛋白
– Inclusion bodies --包涵体
解决途径:高效筛选-标签蛋白
Solution: Efficient screening – tagged proteins
Introduction绪论
Screening challenges 筛选挑战
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Glutathione S-transferase谷胱甘肽转移酶
Maltose binding protein麦芽糖结合蛋白
Histidine(6)组氨酸(6)
Increased selectivity增加选择性
Introduction绪论
Advantages with tagged Protein标签蛋白的优势
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Cell disruption细胞裂解
Analysis分析
Cell cultivation & harvest细胞培养和收取
Expression screening表达筛选
Purification screening纯化筛选
Multiple Constructs多组分
Condition screening条件筛选
1-10 Constructs1-10个组分
Isolation of antibodies
Recover supernatant抗体分离表面回收
Screening workflows 筛选流程Introduction绪论
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SpinTrapTM MultiTrapTM Mag SepharoseTM
50 µl medium/wellWell volume 800 µlManual and robot
100 µl medium/columnVolume 800 µlManual
Variable medium and sample volume
Manual and robot
Formats used in screening筛选用到的产品形式
Introduction绪论
100 µl 填料/柱子800 µl体积
手动
50 µl 填料/孔800 µl体积/孔手动和机器人操作
填料和样品体积可变手动和机器人操作
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ScreeningManual or fully automated protocols
Introduction绪论
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Resinin well
Equilibration
Mix30 sec
Remove the buffer
Sampleaddition
30 min incubation with mixing
Wash (x2)
Elution
Magnetic separator
Resinin well
Equilibration
Mix30 sec
Remove the buffer
Sampleaddition
30 min incubation with mixing
Wash (x2)
Elution
Magnetic separator
Resinin well
Equilibration
Mix30 sec
Remove the buffer
Sampleaddition
30 min incubation with mixing
Wash (x2)
Elution
Magnetic separator
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1 96
Well number
Am
ount
elu
ted
IgG
(µg)
RSD: 4.6 %
Screening with magnetic beadsIntroduction绪论
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Introduction
Conditions screening
His-tagged proteins
Monoclonal antibodies
Refolding conditions for inclusion bodies
Summary
Outline
1. Solubility screening
2. Buffer screening
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Solubility screening
Screen 96 different combinations of buffer, salt, glycerol and reducing agents for a single protein target on His MultiTrapTM FF with a robotic system.
Published by kind permissions from Ruth Steel and Dr. B. L. Grasberger at Johnson and Johnson, Exton, USA
Problem: Histidine tagged NURR1 ligand binding domain (LBD) did not bind using standard protocol. Protein precipitates
Aim: Find the best solubilization and purification conditions to obtain high yield of purified soluble protein
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Increasing NaCl
10% glycerol
5% glycerolβ-mercaptoethanol
Increasing NaCl
10% glycerol
5% glycerolβ-mercaptoethanol
Increasing NaCl
10% glycerol
5% glycerolβ-mercaptoethanol
MES pH 6.0 PIPES pH 6.5
Pi pH 7.0 Pi pH 7.5
HEPES pH 7.5 HEPES pH 8.0
Tris pH 8.0 Tris pH 8.5
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Automated buffer screening simplified the finding of optimal conditions for solubility
Optimized buffer: Tris, pH 8.5, 100 mM NaCl, 10 % glycerol
Results SDS-PAGE
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Scale up on ÄKTAxpressTM using optimized conditions from screening study gave a highly pure protein.
NURR1 is a mixture of monomer, dimer and trimer
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Optimized buffer: Tris, pH 8.5, 100 mM NaCl, 10% glycerol
First step: 1 ml HisTrap FF crude (scale up!)
Second step: HiLoad™ 16/60 Superdex™ 200 pg
System: ÄKTAxpress
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Scale-up
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Aim: Find the best purification conditions for GST-GFP-(His)6
Purification with IMAC is a balance between yield and purity, modulated by the imidazole concentration
Screen 8 different imidazole concentrations and 4 different sample loads in triplicate for a single protein target on His Mag SepharoseTM Ni
His Mag Sepharose Ni™
400 nmSDS-PAGEImageQuant TL
Buffer screening
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M SM FT
Mr x 103
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45
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20.1
14.4
25 % sample load 50 % sample load 75 % sample load 100 % sample load
M
GST-GFP-(His)6
Increasing imidazole0 - 200 mM
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mM imidazole
Purit
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d re
lativ
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(%)
YieldPurity
Optimized buffer:40 mM imidazole with a sample load of 50 % to 100 % of the total binding capacity
Good balance between yield and purity
Results SDS-PAGE
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Outline
Introduction
Conditions screening
His-tagged proteins
Monoclonal antibodies
Refolding conditions for inclusion bodies
Summary
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Screening of elution conditions
Aim: Find the best elution condition for optimal recovery of a monoclonal human IgG expressed in CHO cells
The effect of pH, concentration of NaCl and arginine was studied in a factorial design experiment
Screening of 18 different elution conditions on Protein A Mag SepharoseTM Xtra with MagRack 6
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pH
NaCl
Arg
0M 750 mM
3.0
4.0
0 M
1.0 M
pH
NaCl0M0
1.07504.08
0.53753.59
0.53753.012
0.53754.013
0.503.514
0.57503.515
03753.516
1.03753.517
0.53753.518
1.003.011
1.07503.010
004.07
003.06
0.53753.55
1.004.04
07503.03
07504.02
0.53753.51
Arginineconc. (M)
NaCl(mM)pH
Run order
Experimental design
The cube represents the experimental space according to the experimental design
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Evaluation of study
Optimized buffer: Elute with pH <3.2 for optimal recovery, addition of arginine increases the recovery slightly
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Rec
over
y (%
)
* *
*Precipitation
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Outline
Introduction
Conditions screening
His-tagged proteins
Monoclonal antibodies
Refolding conditions for inclusion bodies
Summary
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AdvantagesHigh expression levels
Simple preparation
Quite pure target protein
Protection from proteolytic enzymes
Expression of toxic proteins
Electron micrograph of E. coliwith inclusion bodiesBy courtesy of Prof. Jonathan King, MIT, Cambridge
Inclusion bodiesIntroductionOutlineInclusion bodies
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Workflows screening
Isolation of inclusion bodies
Cell disruption
Analysis
Cell cultivation & harvest
Expression Screening
Purification screening
Multiple Constructs
Condition screening
1-10 Constructs
Isolation of antibodies
Culture media supernatant
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Workflow refolding
Purification
Refolding
Solubilizationusing denaturants
Analytical techniques for monitoring refolding
Protein refolding
Cell disruption
Freezing/storage
Cell harvest
Inclusion body preparation
Sedimentation and wash
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Additives in refolding buffers
Reduced S-S bridgesDTT, DTE, TCEP, GSHReducing agents
DisruptedDestabilizedUrea Guanidine-HClStrong detergent
Denaturants
Effects
ReducedNeutralUrea (low conc.) Guanidine-HCl (low conc.) Arginine-HCl
Enhanced StabilizedSugarsPolyolsAmmonium sulfateMagnesium chlorideGlycineAlanine
Folding enhancers
ReducedNeutralMild detergentsPEGsProlineCyclodextrins
Aggregation suppressors
Intra- and inter-molecular interactions
Protein structureAdditives
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Refolding techniques
May require affinity tagBinding at denaturing conditions
Fast refolding (<1 hour)High protein concentrationReduced aggregationCombined purification and refolding stepLow buffer consumption
Matrix-assisted refolding
Slow refolding (several hours)Low protein concentrationLarge final volumes
SimpleInexpensive
Dilution/Dialysis
Cons (-)Pros (+)Technique
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Screening strategy
His MultiTrap™ FF
NaCl conc.
Additives
pH/buffersubstance
Stepwise optimization
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DimerEnzyme assay6.172Glucokinase (GLK)
TetramerEnzyme assay5.3464Beta-galactosidase (ß-Gal)
DimerEnzyme assay8.198Citrate synthase (CS)
MonomerEnzyme assay6.235Ferredoxin-NADP+ reductase(FNR)
MonomerFluorescence emission
5.728Enhanced Green Fluorescent Protein (eGFP)
StructureAnalysispI kDaHis-tagged protein
His-tagged 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 Gln Reducing agents (DTT, TCEP)
Buffer + pH
NaCl
Arg + Gln
Additives
Ferredoxin NADP+ reductase
Buffers
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Column: HisTrap™ FF (1ml)Flow rate: 0.5 ml/min
Programming:
Equilibrate with denaturing buffer
Load denatured protein
Refolding buffer
Pause for 20 min
Refolding buffer
Gradient elution
Scale-up: Matrix assisted refolding
histrapfoldingpauseneu2901082009:10_UV1_280nm histrapfoldingpauseneu2901082009:10_Fractions histrapfoldingpauseneu2901082009:10_Logbook
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load refolding
elution of refolded protein
pause
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typical chromatogram
His
-tag
His
-tag
affinity matrix
His
-tag
His
-tag
Structure from the PDP database
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Scale-upScreening
Scale-up
eGFP CS FNR GLK β-gal
Refolding yield (%)
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Monomer 28 kDa
Homodimer 98 kDa
(2x49 kDa)
Homodimer 72 kDa
(2x36 kDa)
Homotetramer 464 kD
(4x116 kDa)
Monomer35 kDa
eGFP CS FNR GLK ß-Gal
% re
fold
ing
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Matrix-assisted refolding
Dilution refolding
Refo
ldin
g yi
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(%)
Refolding yield
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Questions?
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• Increased throughput - Parallel format Shorten the process timeRun more experiments
• Expression screeningQuickly find high producing clones
• Condition screeningRapid protocol optimizationMatrix-assisted refolding
Summary
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MuliTrap, Sepharose, HiTrap, HisTrap, His SpinTrap, His MultiTrap, ÄKTAxpress, ÄKTAexplorer, Mag Sepharose 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.
© 2010 General Electric Company – All rights reserved.First published September 2010.
GE Healthcare Bio-Sciences AB, a General Electric Company.
GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden.
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Thank you for your attention