SHOTGUN MUTAGENESIS MAPPING TECHNOLOGY
Application Notes
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.
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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