Engineering of an anti-epidermal growth factor receptor antibody to single chain format and labeling by sortase A-mediated protein ligation

Madej, Mariusz P., Coia, Gregory, Williams, Charlotte C., Caine, Joanne M., Pearce, Lesley A., Attwood, Rebecca, Bartone, Nick A., Dolezal, Olan, Nisbet, Rebecca M., Nuttall, Stewart D. and Adams, Timothy E. (2012) Engineering of an anti-epidermal growth factor receptor antibody to single chain format and labeling by sortase A-mediated protein ligation. Biotechnology and Bioengineering, 109 6: 1461-1470. doi:10.1002/bit.24407


Author Madej, Mariusz P.
Coia, Gregory
Williams, Charlotte C.
Caine, Joanne M.
Pearce, Lesley A.
Attwood, Rebecca
Bartone, Nick A.
Dolezal, Olan
Nisbet, Rebecca M.
Nuttall, Stewart D.
Adams, Timothy E.
Title Engineering of an anti-epidermal growth factor receptor antibody to single chain format and labeling by sortase A-mediated protein ligation
Journal name Biotechnology and Bioengineering   Check publisher's open access policy
ISSN 1097-0290
0006-3592
Publication date 2012-06
Year available 2011
Sub-type Article (original research)
DOI 10.1002/bit.24407
Open Access Status
Volume 109
Issue 6
Start page 1461
End page 1470
Total pages 10
Place of publication Hoboken, NJ, United States
Publisher John Wiley & Sons
Collection year 2013
Language eng
Formatted abstract
Sortase-mediated protein ligation is a biological covalent conjugation system developed from the enzymatic cell wall display mechanism found in Staphylococcus aureus. This three-component system requires: (i) purified Sortase A (SrtA) enzyme; (ii) a substrate containing the LPXTG peptide recognition sequence; and (iii) an oligo-glycine acceptor molecule. We describe cloning of the single-chain antibody sc528, which binds to the extracellular domain of the epidermal growth factor receptor (EGFR), from the parental monoclonal antibody and incorporation of a LPETGG tag sequence. Utilizing recombinant SrtA, we demonstrate successful incorporation of biotin from GGG-biotin onto sc528. EGFR is an important cancer target and is over-expressed in human tumor tissues and cancer lines, such as the A431 epithelial carcinoma cells. SrtA-biotinylated sc528 specifically bound EGFR expressed on A431 cells, but not negative control lines. Similarly, when sc528 was labeled with fluorescein we observed antigen-specific labeling. The ability to introduce functionality into recombinant antibodies in a controlled, site-specific manner has applications in experimental, diagnostic, and potentially clinical settings. For example, we demonstrate addition of all three reaction components in situ within a biosensor flow cell, resulting in oriented covalent capture and presentation of sc528, and determination of precise affinities for the antibody–receptor interaction.
Keyword scFv
Bioconjugation
Enzyme-mediated ligation
Biosensor
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ
Additional Notes Author post-print permissible.

Document type: Journal Article
Sub-type: Article (original research)
Collections: Non HERDC
Queensland Brain Institute Publications
 
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Created: Thu, 06 Mar 2014, 13:48:39 EST by Rebecca Nisbet on behalf of Clem Jones Centre for Ageing Dementia Research