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Enzymatic functionalization of a nanobody using protein insertion technology.

O Crasson1, N Rhazi1, O Jacquin1

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Protein Engineering, Design & Selection : PEDS
|April 9, 2015
PubMed
Summary

Protein engineering fused a nanobody (cAb-Lys3) to a beta-lactamase enzyme (BlaP), creating a versatile tool. This hybrid protein retains both antibody binding and enzyme activity for novel biotechnological applications.

Keywords:
antibody engineeringmultifunctional enzymenanobodyprotein chimeraβ-lactamase

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Area of Science:

  • Biotechnology
  • Protein Engineering
  • Molecular Biology

Background:

  • Antibody fragments, like nanobodies, are valuable for diagnostics and therapeutics.
  • Developing novel antibody-based products is a priority for biotech and pharmaceutical industries.
  • Modified antibodies, including fragments and conjugates, are emerging tools.

Purpose of the Study:

  • To functionalize a camelid VHH antibody fragment (cAb-Lys3) by inserting it into Bacillus licheniformis beta-lactamase (BlaP).
  • To assess the retained functionality of both the nanobody and the enzyme in the hybrid protein.
  • To explore the utility of this engineered protein in various biotechnological assays and systems.

Main Methods:

  • Protein engineering to create a hybrid protein by inserting cAb-Lys3 into BlaP.
  • Characterization of the hybrid protein's enzymatic activity and antibody binding affinity.
  • Implementation of the functionalized nanobody in enzyme-linked immunosorbent assay (ELISA), biosensor, and drug screening assays.
  • Expression of the hybrid protein on phage surfaces for specific HEWL interaction monitoring.
  • Development of a thrombin-cleavage system for nanobody separation.

Main Results:

  • The engineered hybrid protein retained both BlaP enzymatic activity and cAb-Lys3's nanomolar affinity for HEWL inhibition.
  • The functionalized nanobody was successfully applied in ELISA, potentiometric biosensors, and drug screening.
  • Phage display demonstrated specific HEWL interaction, with beta-lactamase activity serving as a reporter.
  • A system for easy nanobody separation from the carrier protein using thrombin cleavage was established.

Conclusions:

  • Insertion into BlaP is a viable strategy for functionalizing nanobodies.
  • This approach yields versatile tools for innovative biotechnological assays.
  • The developed system enables the creation of easily separable, functionalized nanobody-based tools.