Site-Specific C-Terminal Labeling of Recombinant Proteins with Proximity-Based Sortase-Mediated Ligation (PBSL)

Hejia Henry Wang; Andrew Tsourkas

doi:10.1007/978-1-4939-9546-2_2

Site-Specific C-Terminal Labeling of Recombinant Proteins with Proximity-Based Sortase-Mediated Ligation (PBSL)

Methods Mol Biol. 2019:2012:15-28. doi: 10.1007/978-1-4939-9546-2_2.

Authors

Hejia Henry Wang¹, Andrew Tsourkas²

Affiliations

¹ Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
² Department of Bioengineering, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, PA, USA. [email protected].

PMID: 31161501
DOI: 10.1007/978-1-4939-9546-2_2

Abstract

S. aureus sortase A (SrtA), a calcium-dependent transpeptidase, is frequently employed to site-specifically label the C-terminus of recombinant proteins bearing an LPXTG SrtA recognition motif. Unfortunately, SrtA suffers from low turnover rates, resulting in poor ligation efficiencies even with optimized reaction conditions. In this chapter, we describe proximity-based sortase-mediated ligation (PBSL), which uses the SpyTag-SpyCatcher peptide-protein pair to link SrtA to target proteins and dramatically improves reaction rate and ligation efficiency.

Keywords: Bioconjugation; Proximity ligation; Site-specific labeling; Sortase; SpyCatcher; SpyTag; SrtA.

MeSH terms

Aminoacyltransferases / chemistry*
Aminoacyltransferases / genetics
Bacterial Proteins / chemistry*
Bacterial Proteins / genetics
Cloning, Molecular
Cysteine Endopeptidases / chemistry*
Cysteine Endopeptidases / genetics
Gene Expression
Protein Interaction Domains and Motifs*
Recombinant Fusion Proteins*
Staining and Labeling*

Substances

Bacterial Proteins
Recombinant Fusion Proteins
Aminoacyltransferases
sortase A
Cysteine Endopeptidases