Aldehyde tag

The aldehyde tag is a short peptide tag which can be introduced into fusion proteins and by subsequent treatment with the formylglycine-generating enzyme (FGE) a reactive aldehyde group is generated for further coupling. Since there already is an array of aldehyde-specific reagents commercially available (such as aminooxy or hydrazide reagents), possible applications are diverse and include the conjugation of fluorophores, glycans, PEG (polyethylene glycol) chains or reactive groups for further synthesis (see applications). The aldehyde tag is a short peptide tag which can be introduced into fusion proteins and by subsequent treatment with the formylglycine-generating enzyme (FGE) a reactive aldehyde group is generated for further coupling. Since there already is an array of aldehyde-specific reagents commercially available (such as aminooxy or hydrazide reagents), possible applications are diverse and include the conjugation of fluorophores, glycans, PEG (polyethylene glycol) chains or reactive groups for further synthesis (see applications). The aldehyde tag is an artificial peptide tag recognized by the formylglycine-generating enzyme (FGE). Formylglycine is a glycine with a formyl group (-CHO, an aldehyde) at the α-carbon. The sulfatase motif is the basis for the sequence of the peptide which results in the site-specific conversion of a cysteine to a formylglycine residue. The peptide tag was engineered after studies on FGE recognizable sequences in sulfatases from different organisms. Carrico et al. discovered a high homology in the sulfatase motif in bacteria, archaea as well as eukaryotes. Aldehydes and ketones find use as chemical reporters due to their strong electrophilic properties. This enables a reaction under mild conditions when using a strong nucleophilic coupling partner. Typically, hydrazides and aminooxy probes are used in bioconjugation. They form stabilized addition products with carbonyl groups that are favoured under the physiological reaction conditions. At neutral pH, the equilibrium of Schiff base formation, is lying far to the reactant's side. To make more product named compounds are used to form stable hydrazones and oximes. Since the pH-optimum of 4 to 6 cannot be achieved by adding a catalyst due to associated toxicity, the reaction is slow in live cells. A typical reaction constant is 10−4 to 10−3 M−1 s−1. A carbonyl group is introduced into proteins as a chemical reporter using different techniques, including modern methods like stop codon suppression and the herein discussed aldehyde tag. Limiting the use of aldehydes and ketones is their restricted bioorthogonality in certain cellular environments.Limitations of aldehydes and ketones as chemical reporters are due to Aldehydes and ketones are therefore best used in compartments where such unwanted side reactions are decreased. For experiments with live cells, cell surfaces and extracellular space are typical fielding areas. Nevertheless, a feature of carbonyl groups is the vast number of organic reactions that involve them as electrophiles. Some of these reactions are readily convertible to ligations for probing aldehydes. A rather exotic reaction recently employed for bioconjugation by Agarwal et al. is the adaptation of the Pictet-Spengler-reaction as a ligation. The reaction is known from natural product biosynthetic pathways and has the major advantage that a new carbon-carbon bond is formed. This guarantees long-term stability compared to carbon-heteroatom bonds at same reaction kinetics. The modification of cysteine or, more rarely, serine by FGE is a rather unusual posttranslational modification and was discovered already in the late 1990s. The deficiency of FGE leads to an overall deficiency of functional sulfatases due to a lack of α-formylglycine formation vital for the sulfatases to perform their function. FGE is essential for protein modification and need of high specificity and conversion rate is given in the native setting, which makes this reaction applicable in chemical and synthetic biology. Carrico et al. pioneered the insertion of the modified sulfatase motif peptide into proteins of interest in 2007. Such use of aldehydes and ketones as a chemical reporter in bioorthogonal applications has been applied in self-assembly of cell-lysing drugs, the targeting of proteins, as well as glycans and the preparation of heterobifunctional fusion proteins since then. The formylglycine tag or aldehyde tag is a convenient 6- or 13-amino acids long tag fused to a protein of interest. The 6-mer tag represents the small core consensus sequence and the 13-mer tag the longer full motif.The experiments on the genetically encoded aldehyde tag by Carrico et al. clearly showed the high conversion efficiency with only the core consensus sequence present. Four proteins were produced recombinantly in E.coli with an 86% efficiency of for the full-length motif and >90 % efficiency for the 6-mer determined by mass spectrometry.The size of the sequence is analogous to the commonly used 6x His-Tag and has the advantage that it can also be genetically encoded. The sequence is recognized in the ER solely depending on primary sequence and subsequently targeted by FGE. Notably, in the setup of recombinant expression proteins in E. coli a coexpression of exogenous FGE aids full conversion, although E. coli has endogenous FGE-activity.The introduction of an aldehyde tag as proposed by Carrico et al. has a workflow that consists of three segments: A the expression of the fusion protein, that carries the peptide tag derived from the sulfatase motif, B the enzymatic conversion of Cys to f(Gly) and C the bioorthogonal probing with hydrazides or alkoxy amines (Fig. 1). As seen in Fig. 1, the engineered aldehyde tag consists of six amino acids. A set of organisms from all domains of life was chosen and the sequence homology of the sulfatase motif was determined. The sequence used is the best consensus for sequences found in bacteria, archaea, worms and higher vertebrates.