Total Chemical Synthesis of a Functionalized GFP Nanobody

Chembiochem. 2022 Oct 6;23(19):e202200304. doi: 10.1002/cbic.202200304. Epub 2022 Aug 23.

Abstract

Chemical protein synthesis has proven to be a powerful tool to access homogenously modified proteins. The chemical synthesis of nanobodies (Nb) would create possibilities to design tailored Nbs with a range of chemical modifications such as tags, linkers, reporter groups, and subsequently, Nb-drug conjugates. Herein, we describe the total chemical synthesis of a 123 amino-acid Nb against GFP. A native chemical ligation- desulfurization strategy was successfully applied for the synthesis of this GFP Nb, modified with a propargyl (PA) moiety for on-demand functionalization. Biophysical characterization indicated that the synthetic GFP Nb-PA was correctly folded after internal disulfide bond formation. The synthetic Nb-PA was functionalized with a biotin or a sulfo-cyanine5 dye by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), resulting in two distinct probes used for functional in vitro validation in pull-down and confocal microscopy settings.

Keywords: chemical protein synthesis; click chemistry; nanobodies; protein modifications; solid phase peptide synthesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkynes / chemistry
  • Azides* / chemistry
  • Biotin
  • Click Chemistry
  • Copper / chemistry
  • Disulfides
  • Proteins / chemistry
  • Single-Domain Antibodies*

Substances

  • Alkynes
  • Azides
  • Disulfides
  • Proteins
  • Single-Domain Antibodies
  • Biotin
  • Copper