SHOTGUN MUTAGENESIS TECHNOLOGY
SHOTGUN MUTAGENESIS
Shotgun Mutagenesis is a novel strategy for mapping protein structure-activity relationships 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 functional protein activity.
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HIGH-THROUGHPUT MAPPING OF STRUCTURES
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 a defined function of interest, such as antibody
binding or agonist-induced signaling. Because each clone is sequenced at the
time of library creation, amino acids critical for the function are readily
identified by a loss of reactivity. These residues are mapped onto the protein
structure to visualize functional moieties.
ADVANTAGES OF SHOTGUN MUTAGENESIS
Shotgun Mutagenesis enables
structural analyses of even difficult proteins, such as GPCRs, 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.
Structure-activity relationships 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
Shotgun Mutagenesis has been used
to map diverse GPCR 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 secreted proteins
- Mapping functional regions
of cytoplasmic proteins
- Identifying DNA/RNA active
elements
