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Sortase A Catalyzed Transpeptidation for Protein Bioengineering
Floris J van Dalen1, Hidde L Ploegh2
1Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA. Floris.vanDalen@childrens.harvard.edu.
Methods in Molecular Biology (Clifton, N.J.)
|August 1, 2026
Summary
Sortase A enzyme facilitates protein modification by attaching specific tags. This study demonstrates its use in functionalizing nanobodies (VHHs) with various glycine-based molecules.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Sortase A from Staphylococcus Aureus is a key enzyme for site-specific protein modification.
- It recognizes and cleaves the LPxTG motif, enabling transpeptidation with nucleophiles.
- This method allows for the introduction of diverse functional groups onto proteins.
Purpose of the Study:
- To demonstrate the utility of sortase A for protein functionalization.
- To explore the transpeptidation of nanobodies (VHHs) using sortase A.
- To investigate the use of various oligoglycine nucleophiles for VHH modification.
Main Methods:
- Utilized wild-type and engineered sortase A variants.
- Introduced the LPxTG sortagging motif into nanobody constructs via genetic engineering.
- Synthesized and employed oligoglycine nucleophiles (GGG-nucleophiles) for transpeptidation reactions.
- Performed transpeptidation reactions on two distinct nanobody (VHH) targets.
Main Results:
- Successfully demonstrated sortase A-mediated transpeptidation of nanobodies (VHHs).
- Showcased the versatility of the method with various GGG-nucleophiles.
- Confirmed the efficient modification of VHHs with desired functional groups.
Conclusions:
- Sortase A is a powerful tool for site-specific functionalization of nanobodies.
- The LPxTG motif and oligoglycine nucleophiles provide a flexible platform for protein engineering.
- This approach enables the development of novel VHH-based therapeutics and research tools.
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