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Creating a Selective Nanobody Against 3-Nitrotyrosine Containing Proteins.

Elise M Van Fossen1, Sonia Grutzius1, Carl E Ruby2

  • 1Oregon State University, Department of Biochemistry and Biophysics, Agricultural and Life Sciences, Corvallis, OR, United States.

Frontiers in Chemistry
|March 10, 2022
PubMed
Summary
This summary is machine-generated.

Researchers developed a novel method using genetic code expansion (GCE) to create selective nanobodies (Nbs) targeting oxidized proteins. This advance aids in distinguishing harmful protein modifications in oxidative stress diseases.

Keywords:
genetic code expansionnanobodynitrotyrosineoxidative post translational modificationsingle domain antibody

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunology

Background:

  • Oxidative stress contributes to various pathologies.
  • Identifying specific oxidized protein modifications is crucial for therapeutic development.
  • Current methods for detecting oxidative post-translational modifications (oxPTMs) have limitations.

Purpose of the Study:

  • To develop a technology for creating selective nanobodies (Nbs) against specific oxPTMs.
  • To demonstrate the utility of genetic code expansion (GCE) in producing antigens for Nb selection.
  • To generate an Nb that specifically recognizes a modified protein in the context of oxidative stress.

Main Methods:

  • Utilized genetic code expansion (GCE) to synthesize site-specific 3-nitrotyrosine (nitroTyr) modified 14-3-3 protein.
  • Employed the synthesized modified protein as an antigen for nanobody selection.
  • Screened and characterized nanobodies for selective binding to the nitroTyr modification.

Main Results:

  • Successfully generated a nanobody (Nb) with high selectivity for the 3-nitrotyrosine (nitroTyr) modified 14-3-3 protein.
  • The developed Nb showed reduced recognition of nitroTyr modifications in other protein contexts.
  • This proof-of-concept demonstrates the feasibility of GCE-assisted Nb development for oxPTMs.

Conclusions:

  • Genetic code expansion (GCE) is an effective strategy for producing homogeneous, site-specific oxPTM-containing proteins required for nanobody development.
  • This approach enables the creation of highly specific nanobodies targeting unique oxidative post-translational modifications.
  • The developed anti-nitroTyr Nb and the GCE methodology pave the way for novel diagnostic and therapeutic strategies in oxidative stress-related diseases.