Molecular Affinity Reagents

Brian Kay STA E-mail: bkay@anl.gov     Biographical Sketch Publications

> Research Goals:

• Generate designer affinity reagents
• Map protein-protein interactions  
• Display combinatorial peptides, antibody fragments, & cDNAs on phage and mRNA
• Target validation
• Promote crystallization of proteins  

> Research Summary:                                                                                                    

With the recent completion of sequencing two hundred bacterial and six eukaryotic genomes, we are entering a "post-genomics era". To add value to this accomplishment, the scientific community’s attention is now directed at determining the function of the thousands of gene products in each cell. Traditionally, one valuable type of reagent that is widely used to probe cells and learn when the protein product of a gene is synthesized, where it is localized, and what it is associated with in the cell is affinity reagents. 

To meet the need for generating thousands of affinity reagents, we use phage-display to isolate peptide ligands and antibodies for each protein target. Phage display offers the following conveniences: (1) the peptide or proteins which are expressed on the surface of the viral particles are accessible for interactions with their targets; (2) the recombinant viral particles are stable; (3) the viruses can be amplified, and (4) each viral particle contains the DNA encoding its recombinant genome, thereby providing a physical linkage between the genotype and phenotype. Thus, phage libraries are conveniently screened by isolating viral particles that bind to targets, plaque-purifying the recovered phage, and sequencing the phage DNA inserts. Usually three rounds of affinity selection are sufficient to isolate binding phage.

>>> Phage-display Selection Experiments

Since protein interaction modules bind short peptide sequences, a fruitful approach of identifying their optimal peptide ligands has been to screen combinatorial peptide libraries. Such libraries can be synthesized on a solid support either with mixtures of amino acids or by the “split-recombine” method. Alternatively, phage-displayed combinatorial peptide libraries can be screened with a module and its ligand preferences can be deduced from the primary structures of the selected peptides. Thus, by either approach, one can deduce the optimal ligand preferences of a protein interaction module in a few weeks’ time. Not only is this information useful in understanding how the specificity of modules varies from one another, there is often excellent correspondence between the primary structures of the peptide ligands and regions within known interacting proteins. We have termed this phenomenon “convergent evolution”, and one example is shown below. Hence, a productive process for mapping protein-protein interactions within a proteome is to identify the optimal peptide ligands for a protein interaction module, predict the interacting proteins by computer, and test those hypothetical interactions that make intuitive biological sense.

>>> Peptide ligands for the N-terminal SH3 domain of Crk

 

		phage-display	PxLPxK
		Abl	PLLPTK
		C3G	PALPPK
		Clone S12	PGLPSK
		DOCK	PPLPPK
		Eps15	PPLPPK

Single-chain antibody Fragments of variable regions (scFv) can also be displayed on the surface of bacteriophage M13 and still function in selectively binding their antigens. As described by others, the antigen-binding surfaces of the heavy and light chains can be connected via a 15 amino acid linker, and still bind selectively, and with high affinity, to the original antigen. In addition, such scFv molecules can be tagged at their C-terminus with an epitope (i.e., c-myc) and six histidines, to permit detection and purification, respectively, without affecting the scFv’s ability to bind to antigens. Even though these antibody fragments are much smaller (25 vs. 150 kDa) than the intact molecules, they can bind their antigens with dissociation constants <10 nM. It should also be mentioned that heterodimeric antigen-binding fragments (Fab) can also be displayed on phage particles and are a useful source of affinity reagents. The figure below show an ELISA of phage particles isolated from a scFv library that bind specifically to the GST fusion protein to the ENTH domain of rat epsin. Our goal is to will have the ability to generate antibodies “upon demand”, in two weeks time, to any bacterial, viral, or eukaryotic protein.

ENTH Domain-specific scFv