In Vitro Affinity Screening ofProtein and Peptide Binders by
Megavalent Bead Surface Display
Pietro Gatti-Lafranconi
Dept. of Biochemistry, Univ. of Cambridge (UK)
Malaga, 26 November 2013
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WHY A NEW PROTEIN DISPLAY SYSTEM?
Growing number of formats/scaffolds for molecular recognition
Library screening vs selection
Advantages of an entirely in vitro method
Increasing complexity for applications in binding/catalysis
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Holliger, P. & Hudson, P. J. Nat. Biotechnol. 23, 1126-36 (2005). ; Hosse, R., Rothe, A. & Power, B. Protein Science 15, 14-27 (2006). ; Kutchukian, Yang, Verdine, and Shakhnovich, JACS 131 (13), 4622-4627 (2009) ; Heinis, C., Rutherford, T., Freund, S. & Winter, G. Nat. Chem. Biol. 5, 502-507 (2009).
VARIETY OF FORMATS CALLS FOR FLEXIBLE SCREENING
Natural, designed and chemically-modified protein binders diversity.
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Hoogenboom, H. R. Nat. Biotechnol. 23, 1105-1116 (2005).
AVAILABLE FORMATS, AND THEIR LIMITATIONS
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Hoogenboom, H. R. Nat. Biotechnol. 23, 1105-1116 (2005).
AVAILABLE FORMATS, AND THEIR LIMITATIONS
Mostly in vitro
Display one/few proteins per template
Allow sampling of large libraries
Survival-based selection
I. Selection
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Hoogenboom, H. R. Nat. Biotechnol. 23, 1105-1116 (2005).
AVAILABLE FORMATS, AND THEIR LIMITATIONS
Almost exclusively in vivo
Multiple proteins displayed per template
Small(er) library size
Quantitative screening
II. Screening
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SCREENING COMPROMISES COVERAGE FOR RESOLUTION
experiment 1 experiment 2
62 positives 148 positives
SCREENING COMPROMISES COVERAGE FOR RESOLUTION
experiment 1 experiment 2
from selectionto screening
62 positives 148 positives
31% 29%
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Ideally, we would do this on large libraries (>106) and using small volumes (50 uL per library)
ESSENTIAL FEATURES OF DISPLAY SYSTEMS
--RGYSLGNWVCAAKFESN--
isolate
amplify genotype
accumulate proteinscreen for
binding
decode
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Antje Keppler et al., A general method for the covalent labeling of fusion proteins with small molecules in vivo Nature Biotechnology 21, 86 - 89 (2002)
BUILDING BLOCKS OF BESD
SNAP-tag based genotype/phenotype linkage
magnetic 1-5 um beads water-in-oil emulsion
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Houlihan G, Lowe D, Hollfelder F. SNAP display - an in vitro method for the selection of protein binders. CurrPharm Des. 2013;19(30):5421-8.
BUILDING BLOCKS OF BESD
supportsize (FACS)recovery
covalent linkage genotype/phenotype
monoclonality
SNAP-Display
Diamante, L., Gatti-Lafranconi, P., Schaerli, Y. & Hollfelder, F. In vitro affinity screening of protein and peptide binders by megavalent bead surface display. PEDS (2013).
THE FINAL PRODUCT
Dr. LetiziaDiamante
Quantitative screening after in vitro PCR and protein expression
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MONOCLONAL DNA AMPLIFICATION EFFICIENCY
Amplification of 1 copy of
template DNA in emulsion
leads to 103 – 105 copies bound
to beads, depending on length
and polymerase used.
SATURATION OF DISPLAY LEVELS
• Protein synthesised >> DNA copies
• Excess of anchors bound on beads
• Display levels amplified and normalised
Beads-displayed GFP GFP ‘lost’ in the supernatant
SCREENING OF HA-TAG LIBRARIES
negative
positive
library
1x NNS2x NNS3x NNS
mutant kDDYA 12 nMNYA 18 nMDYS 16 nM
NYS 21 nM
on beads
kD measurement
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SELF-SUFFICIENT AND UN-BIASED
•One single systems toamplify DNAexpress proteinquantify proteinrecover individual DNA variants
•Libraries reduced to individual mutants•Amplification and recovery are unbiased
ePCR
eIVTT
Quantification
1-bead recovery PCR
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MODULAR AND “SIMPLE”
Independent optimisation of:DNA amplificationProtein expressionBinding assay
Required hardwareVortexThermocycler/ThermomixerFACS
polymerase
length
specificity
IVTT extract
Incubation time
Incubation temperature
Presence of cofactors
Concentration of antigen
Competing antigen
• Tags
• GFP
• ScFV
• DARPins