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Epitope Mapping


Shotgun Mutagenesis Overview

Shotgun Mutagenesis is a comprehensive, high-throughput strategy for epitope mapping and protein engineering. Hundreds to thousands of mutations are introduced throughout a target protein and individually tested in human cells by high-throughput screening. The combination of large-scale mutagenesis and rapid cellular testing of natively folded proteins enables us to rapidly map critical domains in structurally complex proteins with a >95% success rate.

  • Automated creation of hundreds to thousands of point mutations covering every residue in a target protein
  • Mutagenesis to alanine, chimeras, or any other desired residue(s)
  • Native expression of each individual mutant in human cells
  • High-throughput testing of each mutant for expression, antibody reactivity, and function

Shotgun Mutagenesis Epitope Mapping

  • Alanine scanning mutagenesis
  • Epitope mapping on GPCRs
  • MPO Toolbox: Better Surface Expression
  • Epitope mapping on viral Envelopes
  • MPO Toolbox: MAb Humanization
  • MPO Toolbox: Higher Expression
  • Successful mapping of 500+ epitopes with > 95% success rate
  • 30+ Publications including Nature, Cell, and PNAS
  • Mapping of complex epitopes including conformational epitopes on GPCRs, ion channels, transporters, viral proteins, and multi-subunit proteins

Value of Shotgun Mutagenesis Epitope Mapping

Antibody Intellectual Property

  • Epitope data provides support for antibody patent requirements
  • Novelty: Epitope data can differentiate novel antibodies from ‘prior art’
  • Non-obviousness: Antibodies with conformational epitopes are usually considered non-obvious and more patentable
  • Written description requirement: High-resolution epitopes using multiple MAbs demonstrate that the inventor is ‘in possession’ of the invention, and support broad patent claims

Antibody Mechanism of Action

  • Epitope data identifies functional regions targeted by antibodies
  • Epitope data can differentiate candidate antibodies for lead selection

Shotgun Mutagenesis Protein Engineering Services: Toolbox™

The MPO Toolbox™ is Integral Molecular’s proprietary strategy for membrane protein and antibody engineering. MPO uses the high-throughput mutagenesis and cellular expression capabilities of Shotgun Mutagenesis to optimize the cell surface expression and conformational stability of membrane proteins, and engineer antibodies for desired qualities. Comprehensive and combinatorial mutagenesis strategies are used to identify variants with improved properties by screening panels of hundreds to thousands of point mutants or chimeras. MPO also utilizes additional strategies for maximal surface expression, such as the inclusion or optimization of signal sequences, chaperones, signaling partners, and trafficking elements. MPO optimizes proteins directly in their host cell, so optimization results are directly translated into manufacturing and expression improvements.

Applications include:

  • Increased cellular expression
  • Improved protein stability
  • Antibody humanization
  • Antibody affinity maturation

Shotgun Mutagenesis

Shotgun Mutagenesis Epitope Mapping and Engineering

Shotgun Mutagenesis is Integral Molecular’s proprietary comprehensive protein structure mapping and engineering platform. Hundreds to thousands of mutations are introduced throughout a target protein and individually tested in human cells by high-throughput screening. The combination of large-scale mutagenesis and rapid cellular testing of natively folded proteins enables us to rapidly identify and engineer critical domains in structurally complex proteins within a fraction of the time conventionally required with a >95% success rate.

Applications include epitope mapping, antibody engineering (affinity maturation and humanization), and protein engineering.

  • Epitope Mapping Services
  • MPO Toolbox™ Protein/Antibody Engineering Services
  • Assay Development and Cell Services

Epitope Mapping Services

The binding site of an antibody, drug, or other protein can be mapped on complex conformational proteins such as GPCRs and viral proteins with greater than 95% success.

Custom epitope mapping services include automated creation of a mutation library (e.g. Ala scan) for a customer-specified target sequence and testing of antibody binding to determine contact residues.

Pre-validated Shotgun Mutagenesis libraries are also available for epitope and other mapping projects, allowing for expedited project turnaround time.

Target Class Therapeutic Area Pre-validated Library
GPCR Oncology CXCR2
Infectious disease Oncology CXCR4
Infectious disease CCR5
Taste perception TAS2R16
Viral Env Infectious disease CHIKV E1/E2
Infectious disease DENV prM/E
Infectious disease HBV sAg
Infectious disease HCV E1/E2
Infectious disease EBOV GP
Infectious disease RSV F
Infectious disease HIV gp41/gp120
Infectious disease Zika prM/E
Cell junction protein Oncology Claudin-1
Oncology Claudin-4
Membrane protein Oncology MCAM
Receptor tyrosine kinase Oncology Her-2
Custom Epitope Mapping Services Available

MPO Toolbox™: Membrane Protein and Antibody Engineering Services

Comprehensive and combinatorial mutagenesis is used to identify protein and antibody variants with desired properties. Prior projects have included optimizing membrane protein surface expression, enhancing the solubility of membrane proteins, humanizing antibodies, and conducting antibody affinity maturation.

Assay Development and Cell Services

Integral Molecular specializes in developing high-throughput cellular assays to assess molecules that modulate receptor function. Prior projects have included high-throughput mutagenesis of chemosensors and assessment of cell signaling responses to small molecules.

Our services include:

  • Assay Development
  • Target Optimization
  • Compound/Antibody Screening
  • Compound/Antibody Characterization

To request a quote, contact us.

Shotgun Mutagenesis FAQs

Q: What is Shotgun Mutagenesis?
A: Shotgun Mutagenesis is Integral Molecular’s proprietary strategy for rapid structure-function analysis. Using high-throughput techniques, every residue in a desired protein is mutated and each mutant protein is analyzed for function in human cells. The effects of each mutation across the target protein are used to map binding sites and engineer the protein.

Q: What kinds of proteins can be tested?
A: Any protein that can be expressed in an animal cell line can be assayed using Shotgun Mutagenesis. Many projects have focused on difficult membrane proteins that correctly express only in human cells, such as GPCRs and viral envelope proteins.

Q: What kinds of assays can be used in Shotgun Mutagenesis?
A: Any cell-based assay that is adapted to microplate format can be used for Shotgun Mutagenesis. Prior projects have commonly included calcium-flux, viral infection, and flow-cytometry-based immunofluorescence assays.

Q: What is the typical turnaround time for a Shotgun Mutagenesis project?
A: Projects timelines are customized for every project based on the starting point of the project, number of mutations made, and assays applied. However, for a typical project, for example, 500 mutations can be made within a month and repeatedly tested for binding or function over the following month.

Q: What is needed to start?
A: Provide us with the sequence of the gene that you would like to study and we will clone, mutate, sequence, verify, and analyze it for you. If you would like the gene analyzed for its interaction with a specific ligand (e.g. antibody, drug, or natural ligand), you will need to provide the ligand.

Q: What is the deliverable from Shotgun Mutagenesis?
A: Deliverables include mutated plasmids, mutant proteins, or a complete report analyzing the data from these mutant proteins, depending on customer needs.