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Combination of ribosome display and next generation sequencing as a powerful method for identification of affibody

Priscillia Lagoutte1, Adrien Lugari1, Céline Elie1

  • 1BIOASTER, 40 Avenue Tony Garnier, 69007 Lyon, France.

New Biotechnology
|January 12, 2019
PubMed

Insights

Researchers developed novel protein binders for CTX-M-15, a key enzyme driving antibiotic resistance in bacteria. This rapid method aids in creating diagnostic and research tools to combat superbugs.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Microbiology

Background:

  • Extended-spectrum β-lactamases (ESBLs), particularly CTX-M-15, are critical drivers of antibiotic resistance in Enterobacteriaceae.
  • CTX-M-15 poses a significant public health threat in both human and animal infections.

Purpose of the Study:

  • To select and characterize novel protein binders targeting the CTX-M-15 enzyme.
  • To establish a rapid method for generating protein ligands for diagnostic and research applications.

Main Methods:

  • Ribosome display technology was employed to select affibody variants from a combinatorial library.
  • Next-generation sequencing (NGS) was used to identify high-affinity binders after iterative selection.
  • Affibody variants were produced, purified, and characterized for stability, affinity, and specificity.

Main Results:

  • Nine affibody variants with high abundance and specific amino acid motifs (QRP, QLH) were identified.
  • Affibodies demonstrated affinities for CTX-M-15 ranging from 0.04 to 2 μM.
  • Selected affibodies recognized CTX-M-15 in various biological contexts, including bacterial lysates, supernatants, and whole bacteria, and modulated enzyme kinetics.

Conclusions:

  • Ribosome display coupled with NGS is an effective strategy for rapid protein ligand generation.
  • The developed affibodies show potential for use in diagnostics and research related to CTX-M-15-mediated antibiotic resistance.
Keywords:
AffibodyBindersCTX-M15NGSRibosome displayβ-Lactamase

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