Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag

Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 03/2009; 106(9):3000-5. DOI: 10.1073/pnas.0807820106
Source: PubMed

ABSTRACT The properties of therapeutic proteins can be enhanced by chemical modification. Methods for site-specific protein conjugation are critical to such efforts. Here, we demonstrate that recombinant proteins expressed in mammalian cells can be site-specifically modified by using a genetically encoded aldehyde tag. We introduced the peptide sequence recognized by the endoplasmic reticulum (ER)-resident formylglycine generating enzyme (FGE), which can be as short as 6 residues, into heterologous proteins expressed in mammalian cells. Cotranslational modification of the proteins by FGE produced products bearing a unique aldehyde group. Proteins bearing this "aldehyde tag" were chemically modified by selective reaction with hydrazide- or aminooxy-functionalized reagents. We applied the technique to site-specific modification of monoclonal antibodies, the fastest growing class of biopharmaceuticals, as well as membrane-associated and cytosolic proteins expressed in mammalian cells.

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Available from: Wenqing Shui, Jun 13, 2015
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    • "Other enzyme mediated chemical tags include the transglutaminase tag (Lin and Ting, 2006), the sortase tag (Popp et al., 2007) and the formylglycine-generating enzyme (FGE) tag (Wu et al., 2009). These labeling approaches have so far been only applicable to cell surface proteins and in vitro tagging. "
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    • "selectively introduced by inserting a CXPXR motif at the site followed by in situ oxidation of the Cys residue in this sequence to a formylglycine group by a coexpressed formylglycine generating enzyme (Carrico et al., 2007; Wu et al., 2009). This approach was successfully applied to site-specific glycosylation of a human growth hormone via oxime formation with an aminooxy-functionalized glycan (Hudak et al., 2011). "
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    • "The technique has been used for the site-specific covalent introduction of fluorophores, affinity tags, and polyethylene glycol chains ( " PEGylation " ) and should also be useful for glycoconjugation (Frese and Dierks 2009). Other possible applications of the formylglycine " aldehyde tag " involve protein immobilization to surfaces for use in microarrays or biosensors or the coupling of specific drugs to protein ligands such as monoclonal antibodies for use in, e.g., tumor targeting (Frese and Dierks 2009; Wu et al. 2009). "
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