china- high throughput screening for tagged...

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

2 /China Seminar Tour 2010

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


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


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


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


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


ScreeningManual or fully automated protocols

Introduction绪论


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


Wash (x2)

Elution

Magnetic separator

Resinin well

Equilibration

Mix30 sec

Remove the buffer

Sampleaddition


Wash (x2)

Elution

Magnetic separator

0

10

20

30

40

1 96

Well number

Am

ount

elu

ted

IgG

(µg)

RSD: 4.6 %

Screening with magnetic beadsIntroduction绪论


Introduction


His-tagged proteins



Summary

Outline

1. Solubility screening

2. Buffer screening


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


Increasing NaCl

10% glycerol

5% glycerolβ-mercaptoethanol

Increasing NaCl

10% glycerol


Increasing NaCl

10% glycerol


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

20

3040506080

20

3040506080

20

3040506080

20

3040506080

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


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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mAU

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Sam

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Inje

ctio

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el F

iltra

tion

A2 A3 A4 A5 A6 A7 A8 A9 A10

A11

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B11

B10 B9

Monomer

Dimer

Trimer

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Sam

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A2 A3 A4 A5 A6 A7 A8 A9 A11

A12

B12

B11

B10 B9

Monomer

Dimer

Trimer

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

A1

A2

A3 A4 A5

A6

A7

A8

A9 A10

A11

A12 B12

B11 B10

Ben

chm

ark

Ben

chm

ark

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A1

A2

A3 A4 A5

A6

A7

A8

A9 A10

A11

A12A1

A2

A3 A4 A5

A6

A7

A8

A9 A10

A11

A12 B12

B11 B10

Ben

chm

ark

Ben

chm

ark

20

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40506080

Scale-up


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


M SM FT

Mr x 103

97

45

66

30

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

0,0

10,0

20,0

30,0

40,0

50,0

60,0

70,0

80,0

90,0

100,0

0 10 20 40 60 80 100

mM imidazole

Purit

y an

d re

lativ

e Yi

eld

(%)

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


Outline

Introduction


His-tagged proteins



Summary


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


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


Evaluation of study

Optimized buffer: Elute with pH <3.2 for optimal recovery, addition of arginine increases the recovery slightly

0

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100

120

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Rec

over

y (%

)

* *

*Precipitation


Outline

Introduction


His-tagged proteins



Summary


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


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


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


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


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


Screening strategy

His MultiTrap™ FF

NaCl conc.

Additives

pH/buffersubstance

Stepwise optimization


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


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


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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mAU

0.0 5.0 10.0 15.0 20.0 25.0 ml

refo

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g

refo

ldin

g

elu

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F2 X1 X2 X3 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12

load refolding

elution of refolded protein

pause

AU

280

nm

typical chromatogram

His

-tag

His

-tag

affinity matrix

His

-tag

His

-tag

Structure from the PDP database


Scale-upScreening

Scale-up

eGFP CS FNR GLK β-gal

Refolding yield (%)

0

20

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60

80

100


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

0

20

40

60

80

100

Matrix-assisted refolding

Dilution refolding

Refo

ldin

g yi

eld

(%)

Refolding yield

31

Questions?


• Increased throughput - Parallel format Shorten the process timeRun more experiments

• Expression screeningQuickly find high producing clones

• Condition screeningRapid protocol optimizationMatrix-assisted refolding

Summary


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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