Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Oct 17;29(10):3245-3249.
doi: 10.1021/acs.bioconjchem.8b00563. Epub 2018 Sep 21.

One-Pot Dual Labeling of IgG 1 and Preparation of C-to-C Fusion Proteins Through a Combination of Sortase A and Butelase 1

Thibault J Harmand  1 , Djenet Bousbaine  1   2 , Alix Chan  3 , Xiaohong Zhang  4 , David R Liu  3 , James P Tam  4 , Hidde L Ploegh  1
Affiliations

One-Pot Dual Labeling of IgG 1 and Preparation of C-to-C Fusion Proteins Through a Combination of Sortase A and Butelase 1

Thibault J Harmand et al. Bioconjug Chem. .

Abstract

Site-specific chemical modification of proteins can assist in the study of their function. Furthermore, these methods are essential to develop biologicals for diagnostic and therapeutic use. Standard protein engineering protocols and recombinant expression enable the production of proteins with short peptide tags recognized by enzymes capable of site-specific modification. We report here the application of two enzymes of orthogonal specificity, sortase A and butelase 1, to prepare non-natural C-to-C fusion proteins. Using these enzymes, we further demonstrate site-selective installation of different chemical moieties at two sites in a full-size antibody molecule.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
C-C protein fusion using sortase A and butelase 1. A) Using a Peg-based linker synthesized by solid phase peptide synthesis (SPPS). The intermediate and the final product were identified by SDS-PAGE and mass spectrometry. [M+H]+ intermediate, calc: 15581.55, obs: 15583.01. [M+H]+ dimer, calc:29905.35, obs: 29908.01 B) Using a double stranded oligonucleotide-based linker. The resulting construct was incubated with EcoRI to cleave the heterodimer as verified by SDS-PAGE.
Figure 2.
Figure 2.
One-pot dual modification of a full-sized antibody using sortase A and butelase 1. The modified heavy and light chains were detected by SDS-PAGE using the appropriate fluorescence excitation and emission channels.
Scheme 1.
Scheme 1.
Sortase A and Butelase 1 ligation mechanisms and our work described in this paper.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Boutureira O, Bernardes GJL (2015) Advances in Chemical Protein Modification. Chem. Rev. 115, 2174–2195. - PubMed
    1. Beckley NS, Lazzareschi KP, Chih H-W, Sharma VK, Flores HL, (2013) Investigation into temperature-induced aggregation of an antibody drug conjugate. Bioconj. Chem. 24,1674–1683. - PubMed
    1. Hamblett KJ, Senter PD, Chace DF, Sun MM, Lenox J, Cerveny CG, Kissler KM, Bernhardt SX, Kopcha AK, Zabinski RF et al. (2004) Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin. Cancer Res. 10, 7063–7070. - PubMed
    1. Kim CH, Axup JY, Schultz PG (2013) Protein conjugation with genetically encoded unnatural amino acids. Curr. Opin. Chem. Biol, 17, 412–419. - PMC - PubMed
    1. Brustad EM, Lemke EA, Schultz PG, Deniz AA (2008) A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer. J. Am. Chem. Soc. 130, 17664–17665. - PMC - PubMed

Publication types

MeSH terms