experimental section hdh - universiteit utrecht
TRANSCRIPT
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Identification and sequencing of VHH against ErbB1, ErbB2 and ErbB3
Experimental section Course Immunobiology
Rob Roovers, Alex Klarenbeek & Hans de Haard
25th of May 2011
Agenda
• Camelid heavy chain antibodies and VHH
• Structure
• Principle library construction
• Phage display based selection
• Selection for function and screening
• Phage display antibody fragments
• Phage morphology
• Replication
• Controlled expression Ab fragments in E coli
• Experimental setup
• Expression of antibody fragments
• Production of phage
Agenda
• Camelid heavy chain antibodies and VHH
• Structure
• Principle library construction
• Phage display based selection
• Selection for function and screening
• Phage display antibody fragments
• Phage morphology
• Replication
• Controlled expression Ab fragments in E coli
• Experimental setup
• Expression of antibody fragments
• Production of phage
Structure of heavy chain antibodies
Combining benefits of antibodies and small chemicals
VH
VLCL
CH1
CH3
CH2
VHH
CH3
CH2VHH
Conventional Antibody
Heavy and light chainsBoth chains required for antigen
binding and stability
Heavy-Chain Antibody
Only heavy chainsFull antigen binding capacity
and highly stable
Ablynx’s Nanobody®®
The smallest functional fragment of a naturally occurring single-chain
antibody
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Identification Ag specific VHH via phage display
pH shock
Phage display based selection on target binding
Selection for function: isolation of antagonistic antibodies
antibodylibrary
Removal unbound phage
Analyze phage antibodies
Elution by competitionwith excess of receptor
Incubate with immobilized cytokineWashing
antibodylibrary
Removal unbound phage
Analyze phage antibodies
Elution by competitionwith excess of receptor
Incubate with immobilized cytokineWashing
400 ng 50 ng 10 ng
TNF-receptor (10 µM)
BSA (10 µM)
Elution
• Conclusion: 20-fold enrichment by elution with receptor
Immobilized TNF
Selection for function: isolation of TNFα antagonistic Nanobodies
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Screening for Lactococcus bacteriophage neutralizing VHH
Agenda
• Camelid heavy chain antibodies and VHH
• Structure
• Principle library construction
• Phage display based selection
• Selection for function and screening
• Phage display antibody fragments
• Phage morphology
• Replication
• Controlled expression Ab fragments in E coli
• Experimental setup
• Expression of antibody fragments
• Production of phage
Phage morphology
• Bacteriophage F1 (M13)
is filamentous phage with
at the tip 3 to 5 copies of
minor coat protein pIII
• The major coat protein
pVIII present in 2,000 to
2,500 copies
• pVII – pIX located at the
other side of the phage
particle
• Bacteriophage F1 (M13) infects E coli by binding to sexpilus (encoded by F episome) via gene 3
• Double stranded (RF) DNA genome replicated within cell
• Single strand DNA packaged into phage particle and released from cells
• No lytic event, E coli cells survive, but grow slower (plaques)
Phage biology: replication cycle
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Expression of functional Fv in periplasm of E coli
Periplasmic expression of Fab in E coli
Phage assembly
• Aminoterminal end gene 3
expressed within periplasm of
E coli, which is mediated by its
own leader sequences
• Compatible with functional
expression of antibody
fragments, i.e. intramolecular
(or canonical) disulfide bridge
formed within each
immunoglobulin domain
• Genome bacteriophage F1
large (6.4 kB) and encodes
structural and non-structural
proteins
• Gene 3 (minor coat protein)
and gene 8 (major coat
protein) used for display
• Origen of replication
responsible for synthesis of
single stranded DNA
Phage genome
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Phagemid based display system
• Phagemid vectors smaller (4.5 kB) then phage vectors and more efficient for cloning of large libraries
• These are plasmids (with antibiotic resistance marker) containing f1 ori
• They only encode gene 3 – antibody fusion
• All other phage proteins (including
wt gene 3) supplied by helperphage (carrying different antibiotic resistance marker)
• Helperphage defective in replication, meaning that produced phage particles mainly contain phagemid
Monovalent versus polyvalent display
Phagemid system used in experimental part
Q
• Phagemid vector derivative of pHEN1, but contains • a hybrid gene3 / pelB leader sequence with SfiI/NcoI cloning site • FR4 with BstEII site • c-myc followed by His6-tag
• Expression in suppressor strain (TG1) results to incorporation of Gln (Q) at amber stop codon within N-terminus gene III in small fraction of translated mRNA; switching to non-suppressor (f.i. TOP F’) gives production of free VHH only
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Phage vector fd-tet-SfiI/NotI
• Phage vector fd-tet-SfiI/NotI also used having a SfiI/NcoI site within hybrid geneIII / pelB leader sequence in combination with a NotI site
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Agenda
• Camelid heavy chain antibodies and VHH
• Structure
• Principle library construction
• Phage display based selection
• Selection for function and screening
• Phage display antibody fragments
• Phage morphology
• Replication
• Controlled expression Ab fragments in E coli
• Experimental setup
• Expression of antibody fragments
• Production of phage
Toxicity of antibody fragments
• Antibody fragments can be very toxic when expressed in E coli • Two phenotypes:
• Induction of cell lysis • Reduction of growth
• During antibody expression or propagation of phage it is VERY IMPORTANT to grow cells into log phase without disturbing effects of toxicity leading to growth advantage of clones expressing non-toxic fragments
Controlled expression by using the Lac promoter
• β-Galactosidase (LacZ) responsible for conversion of disaccharide Lactose into monosaccharides Galactose and Glucose
• If no Lactose, then active inducer binds to operator and thereby blocks transcription of LacZ (panel B)
• By binding of Lactose to inducer prevention of interaction with operator leading to transcription and translation of LacZ (panel A)
A B
Catabolite repression at high Glucose levels
• At low Glucose levels cAMP
concentrations are high, which
binds to Catabolite Activator
Protein (CAP) leading to
association with CAP region on
DNA resulting in high affinity
binding of RNA polymerase to
promoter region and active
transcription of LacZ gene
• At high Glucose concentration
synthesis of cAMP is decreased,
meaning that RNA polymerase
binds with low affinity and
transcription is inhibited
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Production of antibody fragments in E coli • Prepare overnight culture grown in presence of 2% Glucose: NO
ANTIBODY PRODUCTION, thus avoiding stability problems with plasmid; can start from a colony taken from an Amp/Glu plate
• Inoculate 1:100 in fresh medium with Glucose (0.2% according to DeBellis et al or with 2%)
• At late log phase (OD600 = 0.8 – 1.0) spin down cells (for culture grown in 2% Glucose) and resuspend in medium with the inducer IPTG
• When using the DeBellis protocol (0.2% Glucose) simply add IPTG; Glucose is consumed and therefore there is no repression of Lac promoter
• Continue growth and harvest cells
Remark: It is important that cell grow rapidly during the log phase (doubling time of 20 to 30 minutes), meaning that with three hours after starting the culture the OD600 should be reached; if this is not the case, then there is something seriously wrong!!!
DeBellis publication
• Repressor Glucose can be
included at low concentrations
and does not repress during
addition of inducer IPTG
• But if production problems
occur due to productions of
low levels of very toxic
antibody fragments it is
recommended to grow with
2% Glucose and spin away
Glucose during induction
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Phage production phagemid libraries A431 and MCF7
• Library clonal diversity 10E6-10E8 • Implications:
• Inoculum size • Culture size • Rescue of library (i.e. helperphage infection) • Desired phage number to produce for selection
• Phage production in TG1 suppressor strain without inducer • VHH/P3 fusion expression by leaky expression • Efficient display on phage
Library + helperphage à Phage (Φ) particles
(Phagemid AmpR) (KanR)
Production in 2xYT Ampicillin (or Carbenicillin) and Kanamycin NO glucose
Phage production of phagemid libraries
• Experimental procedure • Inoculate library in 2xYT(or LB) media with 2% glucose, and ampicillin • Starting OD600 of culture preferably <0.05 • Grow until early log-phase (OD600=0.5) • Infect with helperphage (= rescue)
• Protocol • 1 OD600 unit TG1 ~ 8EXP8 cells • 50ml of 2xYT 2% glucose, Ampicillin 100ug/ml in sterile baffled flask • Inoculate using 2.5 OD600 units = 2EXP9 cells: Starting OD = 0.05 • Grow until OD600 = 0.5 (time is library dependent) • Infect 2.5ml (1EXP9 cells) with helperphage
• 1:1 helperphage:cells results in 50-80% infection • 10:1 helperphage:cells results in >95% infection
• Incubate for 30min. @37C (without shaking) • Spin culture to remove… glucose! 10min. 4,500 x g @RT • Resuspend cell pellet in 50ml 2xYT medium containing carbenicillin, and
kanamycin • Grow overnight (ON) shaking @37C in baffled flask for phage production
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Phage precipitation and preparation for selection
• Phage precipitation = purification • 2-step procedure
• Preparation for selection • What project? • Antigen QC • Selection “scheme” • Antigen coating
Identification and sequencing of VHH against ErbB1, ErbB2 and ErbB3
Experimental section Course Immunobiology
Rob Roovers, Alex Klarenbeek & Hans de Haard
25th of May 2011