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: 9.67). 03/2009; 106(9):3000-5. DOI: 10.1073/pnas.0807820106
Source: PubMed


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.

Download full-text


Available from: Wenqing Shui, Jun 13, 2015
21 Reads
  • Source
    • "A related concern is the possibility that distribution of dye on the antibody, especially using lysine chemistry, may be different than that which results from solution-based chemistry. However, even with solution-based chemistry , distribution of antibodies with different numbers of dyes per antibody is a major challenge, and there are concerted efforts to develop site-specific conjugation methods (Panowksi et al., 2014; Junutula et al., 2008; Rabuka et al., 2012; Wu et al., 2009). Labeled antibodies often need to be dialyzed into buffer that is appropriate for a downstream application, for example ELISA, internalization studies, cell toxicity assays and others. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Antibodies labeled with small molecules such as fluorophore, biotin or drugs play an important role in various areas of biological research, drug discovery and diagnostics. However, the majority of current methods for labeling antibodies are solution-based and have several limitations including the need for purified antibodies at high concentrations and multiple buffer exchange steps. In this study, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads. High-capacity magnetic antibody capture beads are key to this method and were developed by combining porous and hydrophilic cellulose beads with oriented immobilization of Protein A and Protein G using HaloTag technology. With a variety of fluorophores it is shown that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry. This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications. Copyright © 2015. Published by Elsevier B.V.
    Journal of immunological methods 08/2015; DOI:10.1016/j.jim.2015.08.008 · 1.82 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The pursuit of quantitative biological information via imaging requires robust labeling approaches that can be used in multiple applications and with a variety of detectable colors and properties. In addition to conventional fluorescent proteins, chemists and biologists have come together to provide a range of approaches that combine dye chemistry with the convenience of genetic targeting. This hybrid-tagging approach amalgamates the rational design of properties available through synthetic dye chemistry with the robust biological targeting available with genetic encoding. In this review, we discuss the current range of approaches that have been exploited for dye targeting or for targeting and activation and some of the recent applications that are uniquely permitted by these hybrid-tagging approaches.
    Cell and Tissue Research 03/2015; 360(1). DOI:10.1007/s00441-015-2145-4 · 3.57 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Glycoproteins are an important class of biomolecules involved in a number of biological recognition processes. However, natural and recombinant glycoproteins are usually produced as mixtures of glycoforms that differ in the structures of the pendent glycans, which are difficult to separate in pure glycoforms. As a result, synthetic homogeneous glycopeptides and glycoproteins have become indispensable probes for detailed structural and functional studies. A number of elegant chemical and biological strategies have been developed for synthetic construction of tailor-made, full-size glycoproteins to address specific biological problems. In this review, we highlight recent advances in chemical and chemoenzymatic synthesis of homogeneous glycoproteins. Selected examples are given to demonstrate the applications of tailor-made, glycan-defined glycoproteins for deciphering glycosylation functions.
    Chemistry & biology 01/2014; 21(1):51-66. DOI:10.1016/j.chembiol.2014.01.001 · 6.65 Impact Factor
Show more