Sulfatases and the formylglycine generating enzyme applications in protein engineering

The past decade has provided many exciting advances in the study of biological sulfation. Discovery of the Calpha-formylglycine (FGly) residue in the active site of sulfatases opened new avenues of investigation. Although crucial for sulfatase catalysis, the FGly residue's origins remained unknown until the discovery of the formylglycine generating enzyme (FGE). This work includes an analysis of FGEs' substrate specificity and subsequent application of FGE in a protein engineering context. Additionally, work is presented regarding development of a strategy for sulfatase-based screening and selection.;Chapter 1 provides an overview of investigations and biotechnology uses of FGE. Chapter 2 details the synthesis of a FGly building block suitable for Fmoc-based solid-phase peptide synthesis. An optimization of peptide cleavage conditions was carried out which preceded the successful use of the building block in the synthesis of a peptide derived from a Mycobacterium tuberculosis sulfatase.;Chapter 3 relates the development of a high-throughput assay to probe the substrate specificity FGEs from M. tuberculosis and Streptomyces coelicolor. The enzymes displayed dramatic differences in substrate stringency; these differences were investigated through site-directed mutagenesis. Short peptides, derived from alternate FGE recognition motifs found in the screen, were fused heterologous proteins and expressed in Escherichia coli allowing efficient conversion by E. coli 's endogenous FGE-like activity.;Chapter 4 reports the development of a general strategy for sulfatase-based screening and selection. Using a prodrug-like approach, three caging modules were synthesized and characterized for their abilities to mask the function of other molecules in a sulfatase-dependent manner. This strategy was applied towards identifying the unknown source of FGE-like activity in Caenorhabditis elegans. Additionally, caged luciferin analogues were investigated, revealing species-dependent sulfatase activity profiles.