In:
Biotechnology and Bioengineering, Wiley, Vol. 109, No. 6 ( 2012-06), p. 1461-1470
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. Biotechnol. Bioeng. 2012; 109:1461–1470. © 2011 Wiley Periodicals, Inc.
Type of Medium:
Online Resource
ISSN:
0006-3592
,
1097-0290
Language:
English
Publisher:
Wiley
Publication Date:
2012
detail.hit.zdb_id:
1480809-2
detail.hit.zdb_id:
280318-5
SSG:
12
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