Protein Thioester Synthesis Enabled by Sortase

Abstract

Proteins containing a C-terminal thioester are important intermediates in semi-synthesis. Currently there is one main method for the synthesis of protein thioesters that relies upon the use of engineered inteins. Here we report a simple strategy, utilizing Sortase A, for routine preparation of recombinant proteins containing a C-terminal αthioester. We used our method to prepare two different anthrax toxin cargo proteins: one containing an αthioester and another containing a D-polypeptide segment situated between two protein domains. We show that both variants can translocate through protective antigen pore. This new method to synthesize a protein thioester allows for interfacing of sortase-mediated ligation and native chemical ligation.

Methods for the directed evolution of proteins

“…Owing to the unfavourable kinetics of wild-type SrtA, efficient bioconjugation typically requires equimolar concentration of substrate and enzyme. Iterated rounds of FACS screening with increasing stringency produced evolved variants of SrtA (eSrtA) with 140-fold higher k cat /K m values, enabling new applications [78][79][80][81][82][83] .…”

Immobilization of Actively Thromboresistant Assemblies on Sterile Blood‐Contacting Surfaces

“…[47] We hypothesized that eSrtA may facilitate rapid labeling of TM LPETG with a single azide motif using the commercially available alkylamine derivative NH 2 -PEG 3 -N 3 to generate TM-N 3 (Figure 1a). As shown in Figure 2a, eSrtA dramatically improved the PEGylation efficiency of TM LPETG with 100 molar equiv of 5 kDa NH 2 -PEG probe as compared to WT SrtA, achieving nearly 80% yield after 2 h with 0.1 molar equiv eSrtA.…”

Recent Advances in Chemoenzymatic Bioconjugation Methods

“…The product is then primed for participation in a native chemical ligation reaction, enabling the semisynthesis of, for example, GFP and anthrax toxin. 46 Donnelly and coworkers have used SrtA to introduce a Cu-chelating sarcophagine into an scFv targeting the active conformation of GPIIb/IIIa, a marker of platelet activation. 47 Positron emission tomography (PET) imaging of a murine model of carotid artery thrombosis showed specific uptake of the radiotracer at the site of injury.…”