Engineered reversible inhibition of SpyCatcher reactivity enables rapid generation of bispecific antibodies.

The precise regulation of protein function is essential in biological systems and a key goal in chemical biology and protein engineering. Here, we describe a straightforward method to engineer functional control into the isopeptide bond-forming SpyTag/SpyCatcher protein ligation system. First, we perform a cysteine scan of the structured region of SpyCatcher.

SpyDisplay: A versatile phage display selection system using SpyTag/SpyCatcher technology.

Phage display is an established method for the selection of recombinant antibodies and other proteins or peptides from gene libraries. Here we describe SpyDisplay, a phage display method in which the display is achieved via SpyTag/SpyCatcher protein ligation instead of genetically fusing the displayed protein to a phage coat protein. In our implementation, SpyTagged antibody antigen-binding fragments (Fabs) are displayed via protein ligation on filamentous phages carrying SpyCatcher fused to the pIII coat protein.

Periplasmic expression of SpyTagged antibody fragments enables rapid modular antibody assembly.

Antibodies are essential tools in research and diagnostics. Although antibody fragments typically obtained from in vitro selection can be rapidly produced in bacteria, the generation of full-length antibodies or the modification of antibodies with probes is time and labor intensive. Protein ligation such as SpyTag technology could covalently attach domains and labels to antibody fragments equipped with a SpyTag.