in vitro affinity screening of protein and peptide binders ... · in vitro affinity screening of...

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

Applied Synthetic Biology in Europe26/11/2013 Malaga, Spain

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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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Mostly in vitro

Display one/few proteins per template

Allow sampling of large libraries

Survival-based selection

I. Selection


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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

ACKNOWLEDGEMENTS

Florian Hollfelder

Letizia Diamante

Sylwia Mankowska

Markus Shober

Funding

in vitro affinity screening of protein and peptide binders ... · in vitro affinity screening of...

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