Mark A. Sullivan Structural Genomics of Pathogenic Protozoa Center for Human Genetics and

Application of Phage Antibody Technology to Structural Genomics:

Antibody Selection and Complex Formation

Mark A. SullivanStructural Genomics of Pathogenic

ProtozoaCenter for Human Genetics and

Molecular Pediatric DiseaseUniversity of Rochester

Goal: Develop a panel of scFvs for each protein produced for evaluation in crystallization trials. The scFvs may also be valuable in defining the role of these proteins in future studies of parasite biology.

Ab 1

Ab 4Ab 3

Ab 2

Antibody - Mediated Protein Crystallization

Antibody Facilitated Crystallization

• Majority of purified ORF proteins fail to crystallize• Complexing the targets with an scFv or Fab can

improve the chances of crystallization– Reducing protein flexibility– Providing different surface contacts

• Known structure of antibodies can facilitate solving of structures

• Many antibody variable regions contain multiple methionine residues for selenium incorporation

• May be possible to re-engineer the linker to provide additional methionine residues

Requirements for Demonstrating Feasibility of Phage Display

Technology

• Phage display must yield useful antibodies

• Capable of high throughput isolation• Single-chain Fvs must function

equivalent to Fabs in crystallization• Direct purification of antibody-target

complexes desirable

Essential Elements for Effective Antibody Isolation

• Good library diversity• Stable vector system for efficient selection

– Minimize deletion of scFvs during enrichment

• Good expression and purification of scFvs• Assays for complex formation and effective

purification

~Foldenrichment # distinct scFvs

Enrichment of Library on Leischmania ORFs

WB28 L3223.2 5000 5

EQ37 00822 1000 5

EQ81 00796 500 6

EQ111 00618 100 2

EQ137 00388 <10 none

EQ153 00462 1000 12

EQ165 00076 <10 none

EQ179 00200 100 3

EQ785 00592 1000 3

EQ1747 006539 2000 ND

EQ1963 006877 2000 2

EQ2005 006951 500 4

EQ2175 007234 500 6

EQ2197 007276 1000 2

EQ2213 007307 2000 2

EQ2239 007352 2000 3

Protein ID ORF ID

Immunoaffinity Purification of scFv/Target Complexes on an Anti-Flag Column

+ Ca+2 - Ca+2

The First Complexes - Immunoaffinity Purification of

EQ153-scFv

EQ153/scFv-16 EQ153/scFv-4

Immunoaffinity Purification of scFv/EQ37

EQ37/scFv-5 EQ37/scFv-3

scF

vT

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Loa

dF

TE

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Loa

dF

TE

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con

c.co

mpl

ex

Immunoaffinity Purification of EQ2187 and EQ2005 complexes

EQ2175/scFv-6 EQ2005/scFv-9

scF

vT

arg

et

Loa

dF

TE

lute

scF

vT

arg

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FT

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Unresolved Issues for Complex Preparation

• Effect of reducing agents on scFv stability• Concentration of complex

– Optimum method– Solubility limits of complexes

• Flag epitope accessibility in scFv/target complex

• scFv multimers• scFv suitability vs. Fab

Acknowledgements

• Antibody Lab– Colleen Shea– Laura Bloedorn– Qian Zhao

• SGPP Rochester Solubles– Erin Quartley– Christina DeVries– Julie Babulski– Danielle DeRosa– Eric Phizicky