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• W1997606186 abstract "See accompanying article by Sara Kanje and Sophia Hober DOI: 10.1002/biot.201400808 Chemical modification of an antibody is typically needed for immobilization and functionalization. Targeting the modification to the Fc domain is preferred in this regard in order to preserve antibody-antigen interaction. One rational way to achieve site selectivity is through the C2 domain of protein G that specifically binds to the Fc domain. An antibody can be modified “in situ” by crosslinking C2 that has been engineered to introduce new functionality. The strategy just took a big leap forward with the targeted photocrosslinking technique discussed by Kanje and Hober, who show that C2 purified from bacteria can be crosslinked to Fc through an enzymatically incorporated unnatural amino acid. Modification of antibody for immobilization and detection can be achieved through chemical crosslinking, but the process is inherently stochastic and the extent and location of modification are difficult to control (Fig. 1A). Crosslinking should ideally occur in the Fc region to avoid blocking access to the complementarity determining regions (CDR) of the variable domains [1]. The bacterial protein C2 is useful for targeting Fc, since it binds the domain with high affinity (Fig. 1B). Once C2 is associated with the antibody, it becomes an extension of the antibody, and any modification to the protein is tantamount to a modification to the antibody. This is significant from a practical standpoint because one can engineer an antibody indirectly by engineering an associated C2, which is much smaller in size (∼6 kDa compared to 150 kDa of antibody) and can be expressed in bacteria. Therefore, this strategy has a potential to simplify certain types of antibody engineering to a technology that can be easily performed in bacteria. (A) Chemical labeling leads to non-specific modification (green dots), some of which may interfere with the antibody function. (B) C2 (gray) binds specifically to the Fc domain and can be crosslinked to Fc through engineered BPAs. (C) C2 may be further engineered to include other useful properties, such as targeted biotinylation (triangle). The biotinylated C2 and the conjugated antibody may be immobilized on a streptavidin coated surface. (D) C2 may be engineered instead to contain a fluorescent tag, e.g. GFP (green). The figures are not to the scale. Although C2 binds antibody tightly, covalently linkage between bound C2 and Fc should further solidify its presumed role as a vehicle for surrogate antibody engineering. Bound C2 can be crosslinked to Fc using a photoactivatable crosslinker, although earlier studies either synthesized C2 chemically or only resulted in moderate crosslinking efficiency [2]. In this issue of Biotechnology Journal, Kanje and Hober concurrently simplified the application of the technology and improved its efficiency by biosynthesizing unnatural amino acid-containing C2 and by optimizing the position of the incorporated unnatural amino acid [3]. The engineered C2 contains two photoactivatable amino acids, p-benzoylphenylalanine (BPA), and crosslinks to Fc with 90% efficiency. While the current design optimizes crosslinking to human Fc, the biosynthetic route used for C2 production makes it straightforward to repeat the study for other species. Any modification to covalently linked C2 should be equivalent to directly modifying the Fc domain. The authors demonstrate one such example, in which C2 is genetically fused to a biotin acceptor peptide to allow enzymatic biotinylation by the co-expressed biotin ligase. The antibody-C2 complex can be immobilized on a neutravidin-coated surface through the biotin tag while preserving efficient antigen detection (Fig. 1C). Similarly, the C2-labeled antibody is proficient in detecting an antigen captured on an ELISA plate. It will be interesting to see if the technique is amenable to other types of modification, such as fluorescence modification. For example, it may be possible to obtain a nearly homogenous population of fluorescently labeled antibody if C2 is genetically fused to GFP, which would enable more accurate quantification of the antigen based on fluorescence measurement (Fig. 1D). The current study points to several experimental variations that can be easily tested. For example, biotinylated C2 is currently purified by HPLC because in vivo biotinylation is not complete. Instead, affinity purification on recently reported monomeric streptavidin resin [4] may reduce the effort needed to isolate modified C2, streamlining the workflow even more. There are also other Fc-binding proteins and peptides, some as small as one or two helices [5-7]. Although the binding surfaces somewhat overlap, each protein binds Fc in a unique manner and may thus crosslink differently. The property of the labeled antibody may also depend on the conjugated domain. It would be interesting to compare different crosslinking mechanisms and evaluate their performance. By fusing several venues of protein engineering, Kanje and Hober just opened a treasure trove of new investigative leads. The authors declare no financial or commercial conflict of interest." @default.
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• W1997606186 date "2015-04-01" @default.
• W1997606186 modified "2023-09-26" @default.
• W1997606186 title "More than one way to skin a cat: In-situ engineering of an antibody through photo-conjugated C2 domain" @default.
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• W1997606186 doi "https://doi.org/10.1002/biot.201500051" @default.
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