You are here

Proc Natl Acad Sci U S A DOI:10.1073/pnas.1411179111

Reprogramming the specificity of sortase enzymes.

Publication TypeJournal Article
Year of Publication2014
AuthorsDorr, BM, Ham, HOk, An, C, Chaikof, EL, Liu, DR
JournalProc Natl Acad Sci U S A
Date Published2014 Sep 16
Keywordsalpha-2-HS-Glycoprotein, Aminoacyltransferases, Bacterial Proteins, Biocatalysis, Cysteine Endopeptidases, Directed Molecular Evolution, Humans, Models, Molecular, Mutant Proteins, Staphylococcus aureus, Substrate Specificity

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.


Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID25187567
PubMed Central IDPMC4169943
Grant ListR01 GM065400 / GM / NIGMS NIH HHS / United States
R01 HL056819 / HL / NHLBI NIH HHS / United States
R01 HL56819 / HL / NHLBI NIH HHS / United States
/ / Howard Hughes Medical Institute / United States