Introducing genetically encoded aldehydes into proteins

Abstract

Methods for introducing bioorthogonal functionalities into proteins have become central to protein engineering efforts. Here we describe a method for the site-specific introduction of aldehyde groups into recombinant proteins using the 6-amino-acid consensus sequence recognized by the formylglycine-generating enzyme. This genetically encoded 'aldehyde tag' is no larger than a His(6) tag and can be exploited for numerous protein labeling applications.

N-Myristoyl transferase-mediated protein labelling in vivo

“…A growing range of enzymes have been adapted for use in this approach, including farnesyl transferases, 9,10 biotin ligase 11 and formylglycine-generating enzyme. 12 Despite the great potential of these techniques, 40 their adoption for general labelling applications can be limited by the need for relatively complex synthetic and biochemical protocols.…”

Synthesis of Heterobifunctional Protein Fusions Using Copper‐Free Click Chemistry and the Aldehyde Tag

“…[37] We obtained maximal protein labeling after 24 h at 37 8C (Figure S2 A in the Supporting Information). The reactions of hIgG with a peptide probe, aminooxy-FLAG (AO-FLAG), [32] were dependent on the reagent concentration and required over ten equivalents (100 mm) of AO-FLAG for optimal labeling ( Figure S2B in the Supporting Information). These results highlight the limitations of an exclusively oxime-based conjugation approach with sterically encumbered reactants, and also provided the impetus to explore Cu-free azidealkyne cycloadditions for protein-protein assembly.…”

“…The Cu-free click reactions labeled MBP-fGly more efficiently than treatment with AO-FLAG alone, as demonstrated by immunoblot ( Figure S6 in the Supporting Information). In more detailed comparisons, DIBAC-FLAG reactions were faster at room temperature than the corresponding AO-FLAG reactions at 37 8C and required lower reagent concentrations (Table S2 and To demonstrate the power of the Cu-free click chemistry approach, we generated conjugates of full-length hIgG with hGH [32,34] or MBP (Figure 3 A). These constructs are particularly relevant to ongoing efforts to increase the serum halflife of protein therapeutics (hGH-hIgG) [10,42] or to achieve dual binding specificities in a single molecule (MBPhIgG).…”

“…[32][33][34] The tag consists of a succinct five-residue sequence (CxPxR) that is recognized by the formylglycine-generating enzyme (FGE). FGE oxidizes the genetically-encoded cysteine residue to the aldehyde-bearing residue formylglycine (fGly) during protein expression in either E. coli or mammalian cells (Scheme 1 A).…”

Antibody-drug conjugate library prepared by scanning insertion of the aldehyde tag into IgG1 constant regions

“…Since its introduction more than 10 years ago, 1 the aldehyde tag has become accepted as a convenient, facile method for the site-specific introduction of a bioorthogonal chemical handle into a protein of interest. The chemical handle can be selectively ligated to a compatible linker/payload to yield a defined protein conjugate.…”