Designer Affinity Reagents Brian Kay bkay@uic.edu
Types of Affinity Reagents Src SH3 domain Lysozyme Src SH3 domain FN3 monobody Peptide Ligand Antibody Fragment Scaffold
M13 Bacteriophage 900 nm x 10 nm
Affinity Selection Process
The Fibronectin Type III Domain as a Scaffold Only 94 aa (vs. 250 aa for antibody fragment) Easier to work with than antibody fragments (more stable, express at higher levels in bacteria) Can bind targets inside cells Used by Shohei Koide, Dario Neri, Dane Wittrup, and Rihe Liu as a scaffold for affinity reagent generation Also called monobody
Construction of a Phage-displayed Library of FN3 Monobodies Immunoglobulin-like fold: monobody Small: 94 amino acids Stable: T m = 90ºC BC loop FG loop Expected diversity NNK (5 residues) NNK (5 residues) 1X10 13 (20 10 ) Actual diversity 1.3X10 10 85 electroporations were used to produce 2.8 x 10 10 transformants, 46% of which (1.3 x 10 10 ) have both loops mutated.
Construction of Phage Library by Kunkel Mutagenesis WT-gIII Phagemid DNA Phagemid DNA M13-K07 helper Uracilated ssdna E.coli CJ236 (F dut - ung - ) Phage particle with uracilated DNA Bacterial colonies E. coli TG1 (F dut + ung + ) x x x Heteroduplex x x x Synthesis of heteroduplex dsdna Annealing of mutagenic oligos
Src Family PxxP linker Y SH3 SH2 Kinase domain Create biosensors for members of the Src family of protein tyrosine kinases (54-81% similarity) Fabricate and test biosensors of Src family member activation in living cells SFKs Fgr Fyn Yes Src Lyn Hck Lck Blk
Identification of Lyn SH3 Domain Binders Phage ELISA Phage ELISA OD405nm
Both TA1 and TA8 Are Highly Selective for the Lyn SH3 Domain TA1 TA8
Construction of Secondary Libraries with a Mega-primer Generated by Error-prone and/or Asymmetric PCR 11
Two Variants Bind more than 130-fold Tighter than the Original Clone TA8 2H7 3C12 K D = 6.2 µm K D = 28 nm K D = 47 nm BC loop FG loop WT-TA8: WD APNTQHG YY VT TRPSISK PI 2H7: WD IRNTAHG YY VT TRPKIGL PI 3C12: WD ISNTSHG YY VT TRPKIGL PI The K D s of 2H7 and 3C12 for SH3 domains of Hck and Btk > 3 µm. 12
Successful Pull-down of Lyn from Cells with the Improved FN3 Monobody 13
Abs 405nm Isolated Monobodies Preferentially Bind to the Fyn SH3 Domain in the Src Family
Out of 150 Human SH3 Domains, the G9 Monobody Only Binds Fyn A. B. C. D. Dots lining the bottom and the right side of the membranes are histagged ligands, which are used for the purpose of alignment
ITC Experiments to Determine the Affinity of G9 Monobody to Three SH3 Domains Fyn SH3 Fgr SH3 Yes SH3 K D s: 166 nm ±6 nm N= 0.89 ΔH= 2.22x10 4 cal/mole ΔS= 43.37 Joule/ºK
The SH3 Domain of Src becomes Accessible upon Activation PxxP linker Y SH3 SH2 Kinase domain Inactive Active An affinity reagent that bind to SH3 domain can be used to monitor activation.
Pull-down of Activated Src Akash Gulyani Klaus Hahn (UNC-CH)
Fluorescently labeled Monobody Responds to Src Binding 19
Sensing Src Activation Merocyanine SH3 domain Kinase domain SH2 domain Src kinase
Ratio Imaging Akash Gulyani Klaus Hahn (UNC-CH)
Biosensor Experiments Inject cells with biosensor proteins and monitor changes in fluorescence.
PDGF Induced Dorsal Ruffling Platelet-derived Growth Factor (PDGF) stimulation induces actin-based dorsal protrusions in fibroblasts. Dorsal ruffles are precursors to macropinosomes. Video removed Src activity is previously known to be required for dorsal ruffle formation and macropinocytosis.
Src activation Followed with the Biosensor Video removed Sites of white, red, and yellow coloration are interpreted as sites of Src activation.
Negative Control Video removed Injection of a fluorescent monobody (point mutant) that does NOT bind the Src SH3 domain.
Results of Preliminary Screening External Set 1: Human Proteins Target Full name/function FN3 Hits USP11 Ubiquitin carboxyl-terminal hydrolase 11 GTP-binding protein SAR1a Yes SAR1A Heat shock protein 90kDa beta HSP90B1 member 1 CTBP2 C-terminal-binding protein 2 Yes Phospholipase A-2-activating Yes PLAA protein Ribosomal protein S6 kinase, RPS6KA3 90kDa, polypeptide 3 mitogen-activated protein kinase Yes MAP2K5 kinase 5 CTBP1 C-terminal-binding protein 1 Yes CDK2 Cyclin-dependent kinase 2 Yes MAPK8 mitogen-activated protein kinase 8 Yes SF3A1 Splicing factor 3 subunit 1 Yes COPS5 constitutive photomorphogenic homolog subunit 5 Yes Targets Prepared by the Structural Genomics Consortium (SGC)
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Acknowledgements Renhua Huang Chris Vinci Kevin Gorman Kritika Pershad Funding: NIH U54, Common Fund