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CreHTS® Shotgun Mutagenesis

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For high throughput and high-definition epitope discovery, Creative Biolabs has developed our exclusive CreHTS® Shotgun Mutagenesis Mapping platform. It offers customers a cost-effective service, with comprehensive alanine scanning mutagenesis and high-throughput analysis of user-specified proteins, data collection, and structural analyses. CreHTS® Shotgun Mutagenesis Mapping is a comprehensive, high-throughput strategy for mapping protein structure-activity relationships by evaluating the effects of point mutations across a target protein. Therefore, it can provide customer with valuable and precise information for understanding structural and functional regions of target proteins in amino acid resolution. Moreover, with our extensive experience and rich expertise in protein expressions, our system can also be used for study challenging targets, such as membrane proteins.

Figure 1: Overview of Shotgun Mutagenesis Mapping

Our CreHTS® Shotgun Mutagenesis service involves, but is not limited to:

  1. Custom creation of hundreds to thousands of Ala scan or any other customized substitutions
  2. Comprehensive mutagenesis of every residue in a target protein
  3. High-throughput expression in mammalian cells
  4. Protein characterization within mammalian cells

The first step of this assay is library construction. Every residue in a target protein is systematically substituted to alanine (or any other user-defined amino acid) by automated mutagenesis. Followed by array the entire sequence-verified mutation library in the microplates and used for high-throughput expression in live human cells. Each mutation can then be assayed in parallel for its contribution of binding to antibodies, drugs, or ligands. Therefore, hundreds to thousands of mutations can be constructed and analyzed in the same amount of time. Substitution with alanine residues eliminates side-chain interactions without altering main-chain conformation or introducing steric or electrostatic effects, so is often the preferred choice for testing the contribution of specific side-chains while preserving native protein structure.

CreHTS® Shotgun Mutagenesis can be widely used in:

  1. Antibody epitope mapping
  2. Protein and antibody engineering
  3. Optimization of protein stability or expression
  4. Mapping of functional domains

References:
Eli Berdougo and Benjamin J. Doranz, High-Throughput Alanine Scanning: Epitope Mapping and Engineering Complex Membrane Proteins by Comprehensive Mutagenesis


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