Abstract
To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields. After further investigation, we confirmed that both aerobic prokaryotic (Streptomyces coelicolor) and eukaryotic (Homo sapiens) FGEs contain a copper cofactor. The complete kinetic parameters for both forms of FGE are described, along with a proposed mechanism for FGE catalysis that accounts for the copper-dependent activity.
Highlights
Aerobic formylglycine-generating enzyme (FGE) converts cysteine to formylglycine in vivo
In Vitro Conversion of Intact monoclonal antibody (mAb) with FGE—Hs-cFGE was added to a solution of IgG containing the aldehyde tag in 25 mM TEAM, pH 9.0, with 50 mM NaCl and 1 mM ME
FGEs from two species, S. coelicolor and H. sapiens, were selected for study because they had been previously characterized to some extent [7,8,9], and because they represented both prokaryotic and eukaryotic forms of the enzyme
Summary
Aerobic formylglycine-generating enzyme (FGE) converts cysteine to formylglycine in vivo. In parallel work described here, we explored the value of performing conversion of Cys to fGly in vitro with purified recombinant FGE (Fig. 1b) This goal requires an efficient enzyme that is stable during the course of a biocatalytic reaction. Our initial experiments employed a 14-amino acid peptide substrate, with which we determined the optimal conditions for in vitro conversion This model system allowed us to explore the mechanism through which FGE converts Cys to fGly. Researchers currently propose a mechanism for FGE that does not require an exogenous cofactor for oxygen activation or catalysis. By defining a biocatalytic protocol for in vitro conversion of Cys to fGly, we enable a straightforward, reliable way to install a sitespecific, bioorthogonal functional group on any folded protein These abilities mutually reinforce the unique utility of FGE for aldehyde production in vivo and in vitro
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