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. 2008 Sep 17;130(37):12240-1.
doi: 10.1021/ja804530w. Epub 2008 Aug 23.

New aldehyde tag sequences identified by screening formylglycine generating enzymes in vitro and in vivo

Jason S Rush  1 , Carolyn R Bertozzi
Affiliations

New aldehyde tag sequences identified by screening formylglycine generating enzymes in vitro and in vivo

Jason S Rush et al. J Am Chem Soc. .

Abstract

Formylglycine generating enzyme (FGE) performs a critical posttranslational modification of type I sulfatases, converting cysteine within the motif CxPxR to the aldehyde-bearing residue formylglycine (FGly). This concise motif can be installed within heterologous proteins as a genetically encoded "aldehyde tag" for site-specific labeling with aminooxy- or hydrazide-functionalized probes. In this report, we screened FGEs from M. tuberculosis and S. coelicolor against synthetic peptide libraries and identified new substrate sequences that diverge from the canonical motif. We found that E. coli's FGE-like activity is similarly promiscuous, enabling the use of novel aldehyde tag sequences for in vivo modification of recombinant proteins.

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Figures

Figure 1
Figure 1
Reaction catalyzed by FGE.
Figure 2
Figure 2
A high-throughput assay for FGE activity.
Figure 3
Figure 3
FGE activity on peptide substrates. (a) Relative activity of S. coelicolor (blue) and M. tuberculosis (red) FGEs on peptides derived from the S. coelicolor sulfatase motif. (b) Relative activity of S. coelicolor (blue) and M. tuberculosis (red) FGEs on peptides derived from the M. tuberculosis sulfatase motif. Error bars represent the standard deviation of three replicates.
Figure 4
Figure 4
Models of prokaryotic FGE active sites with peptide substrate bound. (a) Crystal structure of S. coelicolor FGE with modeled peptide substrate. (b) Homology model of M. tuberculosis FGE with modeled peptide substrate. The substrate peptide shown in cyan is CTPSR. Colors indicate electrostatic potential (blue, positive; red, negative).
Figure 5
Figure 5
SDS-PAGE of MBP constructs bearing the C-terminal aldehyde tag sequences shown above each lane. The proteins were expressed in E. coli, purified on Ni-NTA spin columns, and reacted with Alexa Fluor 647 C5-aminooxyacetamide (Aminooxy Alexa Fluor 647). Fluorescence images of the gel are shown. Top, Alexa Fluor 647. Bottom, protein loading as determined by Sypro Orange.
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