Integral Molecular

SHOTGUN MUTAGENESIS MAPPING TECHNOLOGY

SHOTGUN MUTAGENESIS MAPPING
Shotgun Mutagenesis is a novel strategy for epitope mapping by rapidly evaluating functional effects of point mutations across an entire target protein. Using a patented high-throughput expression method, thousands of point mutations of a target protein can be concurrently evaluated for antibody binding, all at a fraction of the cost of traditional site-directed mutagenesis.

HIGH-THROUGHPUT EPITOPE MAPPING
Shotgun Mutagenesis begins with the creation of a customized plasmid library for a target gene, each clone in the library bearing a unique amino acid mutation. Clones are individually arrayed in microplates and expressed within living mammalian cells. Each clone is validated for expression on the cell surface and for full-length translation to identify the most useful clones containing isolated amino acid substitutions. The entire library of clones is then simultaneously tested for antibody binding or other functional activities. Because each clone is sequenced at the time of library creation, amino acids critical for the epitope are readily identified by a loss of reactivity. These residues are mapped onto the protein structure to visualize epitope maps.

ANTIBODY HUMANIZATION
Shotgun Mutagenesis technology can be used to germline-humanize therapeutic monoclonal antibodies (MAbs) in order to minimize immunogenicity and increase efficacy. This strategy involves comprehensive substitution of every amino acid residue within the variable chains (including CDRs) that diverges from consensus human germline sequences. Antibody variants are assessed for binding to comprehensively define all residues that can tolerate humanization without a loss in activity. Human cellular expression of antibodies in this system ensures proper folding and post-translational modifications, as well as production of whole IgG molecules to assay antibodies directly in their final format.

ADVANTAGES OF SHOTGUN MUTAGENESIS MAPPING
Shotgun Mutagenesis enables mapping of even difficult proteins, such as GPCRs and viral envelopes, that require eukaryotic cells for proper expression, folding, and function, and whose structures cannot be routinely analyzed by direct methods such as crystallography or NMR. Maps determined by conventional site-directed mutagenesis can require years to map even fractions of a protein’s topography. By comparison, Shotgun Mutagenesis enables comprehensive analysis of every amino acid in the protein within weeks of initiating an experiment. Expressed libraries can be analyzed using any microplate-based cellular assay, allowing diverse protein interactions and functions to be mapped. Integral Molecular has developed customized software for tracking and analyzing the tens of thousands of data points involved in array production, validation, and experimentation.

APPLICATIONS OF SHOTGUN MUTAGENESIS MAPPING
Shotgun Mutagenesis has been used to map diverse protein interactions, including the binding sites for antibodies, agonists, and small molecules. Antibody epitopes and drug binding pockets can be mapped to specific amino acids and, as desired, to specific atoms within amino acid side chains. These maps have direct utility for understanding the structural and functional regions of proteins, for protein modeling and in silico docking, and for making lead candidate and intellectual property decisions. Additional applications of Shotgun Mutagenesis include:

Identifying membrane protein signaling motifs
Optimizing the activity of antibodies and secreted proteins
Mapping functional regions of cytoplasmic proteins
Identifying DNA/RNA active elements