John M Antos

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Genetically encoded reporter constructs that yield fluorescently labeled fusion proteins are a powerful tool for observing cell biological phenomena, but they have limitations. Sortagging (sortase-mediated transpeptidation) is a versatile chemoenzymatic system for site-specific labeling of proteins with small (<2 kDa) probes. Sortagging combines the(More)
A general chemoenzymatic method for the site-specific attachment of lipids to protein substrates is described. Sortase A is used to append short lipid-modified oligoglycine peptides to the C terminus of protein substrates bearing a five amino acid sortase A recognition sequence (LPETG). We demonstrate the attachment of a range of hydrophobic modifications(More)
The unique reactivity of two sortase enzymes, SrtA(staph) from Staphylococcus aureus and SrtA(strep) from Streptococcus pyogenes, is exploited for site-specific labeling of a single polypeptide with different labels at its N and C termini. SrtA(strep) is used to label the protein's C terminus at an LPXTG site with a fluorescently labeled dialanine(More)
We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ~12% of the(More)
Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and(More)
Creation of functional protein bioconjugates demands methods for attaching a diverse array of probes to target proteins with high specificity, under mild conditions. The sortase A transpeptidase enzyme from Staphylococcus aureus catalyzes the cleavage of a short 5-aa recognition sequence (LPXTG) with the concomitant formation of an amide linkage between an(More)
The broad utility of protein bioconjugates has created a need for new and diverse strategies for site-selective protein modification. In particular, chemical reactions that target alternative amino acid side chains or unnatural functional groups are emerging as a valuable complement to more commonly used lysine- and cysteine-based strategies. Considering(More)
Protein array technology is an emerging tool that enables high-throughput screening of protein-protein or protein-lipid interactions and identification of immunodominant antigens during the course of a bacterial or viral infection. In this work, we developed an Influenza virus protein array using the sortase-mediated transpeptidation reaction known as(More)
A new transition metal-based reaction has been developed for the selective modification of tryptophan residues on protein substrates. After activation of vinyl-substituted diazo compounds by Rh2(OAc)4, the resulting metallocarbenoid intermediates were found to modify indoles in aqueous media despite competing reactions with water. Both N- and 2-substituted(More)
A modified sortase A recognition motif containing a masked Ni(2+)-binding peptide was employed to boost the efficiency of sortase-catalyzed ligation reactions. Deactivation of the Ni(2+)-binding peptide using a Ni(2+) additive improved reaction performance at low to equimolar ratios of the glycine amine nucleophile and sortase substrate. The success of this(More)