Power to the protein: enhancing and combining activities using the Spy toolbox

Abstract

<p>A peptide with simple and selective reactivity expands the function of proteins, from single molecule analysis to potential clinical application.</p>

Overcoming Symmetry Mismatch in Vaccine Nanoassembly through Spontaneous Amidation

“…[9] SpyCatcher is a1 2kDa protein that we previously engineered to form aspontaneous amide bond to its peptide partner SpyTag ( Figure 1A). [10] While the display of monomeric antigens using modular ligation ( Figure 1B)i se stablished, [11][12][13] there has been no systematic study of the display of antigens possessing different cyclic or dihedral symmetries ( Figure 1C). However,alarge fraction of clinically important antigens are multimeric,i ncluding surface proteins from influenza, Ebola virus and coronaviruses.…”

Overcoming Symmetry Mismatch in Vaccine Nanoassembly through Spontaneous Amidation

“…[35,36] In contrast to VLPs,a nti-platform responses are problematic where cell infection is required for vaccine function, for example,adenoviral vectors. [37] Pre-existing human antibodies against the orthogonal SnoopTag/SnoopCatcher pair were detected in only 25 %o fp eople.T herefore,S noopTagmediated assembly in heterologous prime-boost immunization is an option, if repeated use of SpyCatcher003-mi3 is ever problematic.T ag/Catcher conjugation has been applied for decoration of CAR-T cells and in vivo imaging, [10] where the consequences of pre-existing immune responses for clinical development are likely to be more important.…”

“…SpyTag-mediated amidation has been tested in arange of pre-clinical vaccine models,p articularly for malaria, [9] but it has not been explored to what extent people have pre-existing antibodies against SpyTag/SpyCatcher.S pyTag/SpyCatcher was engineered from the CnaB2 domain of the FbaB adhesion protein from the common human pathogen Streptococcus pyogenes. [10] S. pyogenes causes infection in 700 million people worldwide per year. [19] FbaB is predominantly expressed by the M3 serotype of S. pyogenes.…”

Periplasmic Expression of SpyTagged Antibody Fragments Enables Rapid Modular Antibody Assembly

“…The SpyTag technology has been successfully adopted for a broad range of applications due to its simple, fast, robust, and quantitative protein ligation reaction 14,15 . In this study, we show that all published variants of the SpyTag are prone to cleavage by proteases when expressed in the periplasm of E. coli.…”

“…For clarity, we will be using the terms SpyTag1/SpyCatcher1 when referring specifically to the original SpyTag/SpyCatcher protein sequences and will use the unnumbered names as generic terms. The versatility of this system was shown in many publications in a broad range of different applications and has been the subject of several review articles 14,15 .…”