Introducing Cysteines into Nanobodies for Site-Specific Labeling

Simon Boje Hansen; Kasper Røjkjær Andersen

doi:10.1007/978-1-0716-2075-5_16

Introducing Cysteines into Nanobodies for Site-Specific Labeling

Methods Mol Biol. 2022:2446:327-343. doi: 10.1007/978-1-0716-2075-5_16.

Authors

Simon Boje Hansen¹, Kasper Røjkjær Andersen²

Affiliations

¹ Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark.
² Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark. kra@mbg.au.dk.

PMID: 35157281
DOI: 10.1007/978-1-0716-2075-5_16

Abstract

We have developed a generally applicable methodology for cysteine mutagenesis of nanobody (Nb) framework region serine residues. This strategy allows for subsequent labeling with thiol-reactive compounds without disrupting Nb antigen binding. We provide a protocol for production, labeling, and affinity determination of cysteine-engineered Nbs (cys-Nbs) with Alexa Fluor 488-maleimide and the mercury compound para-chloromercuribenzoic acid (PCMB). Alexa Fluor 488- and PCMB-labeled cys-Nbs can be used for immunofluorescence microscopy and experimental phasing in crystallography, respectively.

Keywords: Cysteine; Experimental phasing; Imaging; Maleimide; Mercury; Nanobody; Site-specific labeling.

MeSH terms

Cysteine / chemistry
Fluoresceins
Serine
Single-Domain Antibodies* / chemistry
Sulfonic Acids

Substances

Fluoresceins
Single-Domain Antibodies
Sulfonic Acids
alexa fluor 488
Serine
Cysteine