Sortase-Mediated Conjugation: Protein & Antibody Engineering
Sortase conjugation enables precise protein and biologic engineering, enhancing therapeutic performance and overcoming traditional method limits. BPS Bioscience offers kits and services to support your protein labeling.
Sortase-Mediated Conjugation: A Precise Tool for Protein & Antibody Engineering
Sortase-mediated conjugation is a powerful technique for the precise engineering of enzymatic tools or therapeutic biologics. It enables site-specific attachment of antibodies and proteins to various molecules and materials, including fluorescent labels for imaging and sorting, pharmaceutical compounds, nanoparticles, and even cells for drug delivery. This enzymatic approach overcomes the limitations of conventional chemical conjugation methods, which can be random and compromise antibody function.
Applications of Sortase-mediated engineering include Fc-oriented antibody labeling to preserve antigen recognition, PEGylation to extend protein half-life while maintaining biological activity, generation of circular polypeptide for increased thermal stability, and enzyme immobilization for industrial applications.
A major use of Sortase-mediated conjugation is in the field of antibody-drug conjugates (ADCs), where it ensures site-specific payload attachment, avoiding the heterogeneity seen in traditional methods. Indeed, by introducing a Sortase recognition sequence at a defined site on the antibody, researchers achieve consistent drug-to-antibody ratios, improving reproducibility and therapeutic efficacy. The reaction occurs under mild conditions, preserving the antibody’s structure and function.
Mechanism of Sortase A-mediated Conjugation
Sortase A, a well-characterized bacterial transpeptidase from Staphylococcus aureus, naturally attaches proteins to bacterial cell walls, performing a critical function in bacterial virulence. It specifically recognizes the LPXTG sequence, cleaving between threonine (T) and glycine (G). The enzyme contains a cysteine residue in its active site, which forms a thioacyl intermediate that can immediately react with an oligo-glycine nucleophile, covalently linking the protein substrate to the oligo-glycine.
Biotechnology harnesses this specificity to precisely label or modify virtually any protein of interest in solution or on cell surfaces, enabling applications in biologic engineering, therapeutics, and diagnostics.
To label a protein of interest:
Insert an LPXTG sequence into the protein of interest at cloning
Add Sortase A and a triglycine-linked label (for example, a fluorophore) or payload to the purified recombinant protein
Advantages
Precise labeling: highly specific to the LPXTG sequence, cleaves between T/G and can attach a poly-G peptide to T, minimizing disruption of protein function.
Versatility: any fluorophore, peptide, or substrate containing a poly-Glycine can be attached to the target protein or antibody.
Allows for in situ or in vivo protein labeling or modification.
Mild reaction conditions minimize risks of denaturation or degradation.
High efficiency >90%, resulting in high yields of conjugated target protein.
Mutants of S. aureus Sortase A may display improved efficiency toward certain substrates, therefore, Sortase mutant sampling may be performed for method optimization.
Engineered Sortase variants now overcome previous limitations in activity and stability, broadening their use in therapeutics, biorthogonal chemistry, biomaterials, and biosensors. This versatile methodology offers a robust and reproducible approach that enables both targeted delivery systems and enhanced protein properties. BPS Bioscience provides multiple options for Sortase-based protein and antibody conjugation.
SiMPLe CHC Antibody Labeling Kit
The Antibody Labeling Kit (BPS Bioscience #82155) achieves homogeneous labeling of antibodies containing sequence LPXTG at the C-terminus of the heavy chain. Poly-glycine-containing molecules (fluorophores, biotin, peptides) are compatible with this kit. The site-specific conjugation ensures that the antigen-binding site remains available. The kit is ideal for ADCs or flow cytometry antibodies.
SiMPLe Protein Labeling Kit
The Protein Labeling Kit (BPS Bioscience #79392) achieves direct conjugation of any LPXTG-containing protein with an efficiency greater than 90%. This kit relies on the highly active Sortase 5Δ Pentamutant for high yield and contains purification columns, and Ubiquitin-LPETGH6 and GGG-Clover as positive controls.
Recombinant Sortase Variants & Sampling kit
The Sampling kit provides wildtype Sortase A and four different mutants to evaluate which is best for a chosen application.
Mazamanian, S.K., Liu, G., Ton-That, H. & Schneewind, O. Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285, 760-763 (1999).
Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128-136 (2008).
Stephanopoulos, N. & Francis, M.B. Choosing an effective protein bioconjugation strategy. Nat. Chem. Biol. 7, 876-884 (2011).
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