Reprogramming the specificity of sortase enzymes.
نویسندگان
چکیده
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.
منابع مشابه
Structural Basis for Group B Streptococcus Pilus 1 Sortases C Regulation and Specificity
Gram-positive bacteria assemble pili through class C sortase enzymes specialized in polymerizing pilin subunits into covalently linked, high-molecular-weight, elongated structures. Here we report the crystal structures of two class C sortases (SrtC1 and SrtC2) from Group B Streptococcus (GBS) Pilus Island 1. The structures show that both sortases are comprised of two domains: an 8-stranded β-ba...
متن کاملEngineering the substrate specificity of Staphylococcus aureus Sortase A. The beta6/beta7 loop from SrtB confers NPQTN recognition to SrtA.
The Staphylococcus aureus transpeptidase Sortase A (SrtA) anchors virulence and colonization-associated surface proteins to the cell wall. SrtA selectively recognizes a C-terminal LPXTG motif, whereas the related transpeptidase Sortase B (SrtB) recognizes a C-terminal NPQTN motif. In both enzymes, cleavage occurs after the conserved threonine, followed by amide bond formation between threonine ...
متن کاملSortase-catalyzed transformations that improve the properties of cytokines.
Recombinant protein therapeutics often suffer from short circulating half-life and poor stability, necessitating multiple injections and resulting in limited shelf-life. Conjugation to polyethylene glycol chains (PEG) extends the circulatory half-life of many proteins, but the methods for attachment often lack specificity, resulting in loss of biological activity. Using four-helix bundle cytoki...
متن کاملCrystal Structure of the Streptomyces coelicolor Sortase E1 Transpeptidase Provides Insight into the Binding Mode of the Novel Class E Sorting Signal
Many species of Gram-positive bacteria use sortase transpeptidases to covalently affix proteins to their cell wall or to assemble pili. Sortase-displayed proteins perform critical and diverse functions for cell survival, including cell adhesion, nutrient acquisition, and morphological development, among others. Based on their amino acid sequences, there are at least six types of sortases (class...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- Proceedings of the National Academy of Sciences of the United States of America
دوره 111 37 شماره
صفحات -
تاریخ انتشار 2014