Reprogramming the specificity of sortase enzymes

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 fluorophoreprotein-PEG conjugates for two therapeutically relevant fibroblast growth factor proteins (FGF1 and FGF2), and the orthogonal conjugation of fluorescent peptides onto surfaces.protein evolution | enzyme specificity

Methods for the directed evolution of proteins

“…The development of a negative screen (also known as counterscreen) using unlabelled competitor substrates enabled our laboratory 84 to evolve reprogrammed orthogonal sortases that selectively conjugate LAETG or LPESG substrates. Because substrates are applied ex vivo, this approach is not limited to genetically encoded peptide substrates, and it should be possible to design similar screens for enzymes that catalyse many different classes of bond-forming reactions.…”

An Apoptosis‐Inducing Peptidic Heptad That Efficiently Clusters Death Receptor 5

“…Fort his purpose we applied engineered sortases referred to as SrtA1-3 (Table S1), which catalyze formation of an amide bond between glycine and b-hydroxy-bearing amino acids (Ser, Thr) located, respectively,a tt he N-terminal oligoglycine and the Cterminal LX i EX ii Gc ounterparts (Scheme 2, Figure S2). [15] Theu sed sortases were tailored to recognize certain peptide tags enabling modularity of ligand attachment (Section S1.6, Figure S2, S3, and Table S2). Thus,t he Fc scaffold 4 was designed to carry two payloads either C-o rN-terminally (conjugates 9, 10,F igure 2, Table S3), and 4payloads (two at the C-a nd two at the N-terminus;c onstruct 11,F igure S3).…”

Recent advances in enzyme promiscuity

“…Recently, two orthogonal sortase A variants have been developed by applying directed laboratory evolution technique that recognize each of two different substrates, LAXTG and LPXSG, with high activity and specificity. The evolved sortases exhibit changes in specificity up to 51,000-fold, relative to the starting sortase without much loss of catalytic activity [46].…”