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Proc Natl Acad Sci U S A. 2014 Sep 16;111(37):13343-8. doi: 10.1073/pnas.1411179111. Epub 2014 Sep 3.

Reprogramming the specificity of sortase enzymes.

Author information

1
Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; and.
2
Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, and the Wyss Institute of Biologically Inspired Engineering at Harvard University, Boston, MA 02115.
3
Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; and drliu@fas.harvard.edu.

Abstract

Staphylococcus aureus sortase A catalyzes the transpeptidation of an LPXTG peptide acceptor and a glycine-linked peptide donor and has proven to be a powerful tool for site-specific protein modification. The substrate specificity of sortase A is stringent, limiting its broader utility. Here we report the laboratory evolution of two orthogonal sortase A variants that recognize each of two altered substrates, LAXTG and LPXSG, with high activity and specificity. Following nine rounds of yeast display screening integrated with negative selection, the evolved sortases exhibit specificity changes of up to 51,000-fold, relative to the starting sortase without substantial loss of catalytic activity, and with up to 24-fold specificity for their target substrates, relative to their next most active peptide substrate. The specificities of these altered sortases are sufficiently orthogonal to enable the simultaneous conjugation of multiple peptide substrates to their respective targets in a single solution. We demonstrated the utility of these evolved sortases by using them to effect the site-specific modification of endogenous fetuin A in human plasma, the synthesis of tandem fluorophore-protein-PEG conjugates for two therapeutically relevant fibroblast growth factor proteins (FGF1 and FGF2), and the orthogonal conjugation of fluorescent peptides onto surfaces.

KEYWORDS:

enzyme specificity; protein evolution

PMID:
25187567
PMCID:
PMC4169943
DOI:
10.1073/pnas.1411179111
[Indexed for MEDLINE]
Free PMC Article

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