Cyclization tag for the detection and facile purification of backbone-cyclized proteins

Backbone-cyclized proteins, with their characteristic stability toward denaturants such as heat and chemicals, are becoming increasingly significant in many applications. Intein-mediated protein cyclization is the most efficient and frequently used method of choice and has been successfully applied to various targets, achieving stable proteins. However, the detection and isolation of the cyclic protein from the linear one after cyclization is very difficult because the backbone-cyclized protein and the linear one (a by-product formed during the cyclization reaction), which originated from the same molecule, are almost identical in terms of their size. Thus, we first developed a split c-myc tag system; the active c-myc tag was formed only in the backbone-cyclized protein and not in the linear by-product from the inactive precursor, and this helps both the detection and purification of the backbone-cyclized proteins. This tag system, which we called a cyclization tag, was further engineered in its sequence to develop an engineered c-myc (e-myc) tag with enhanced efficiency in the backbone cyclization reaction while keeping its specificity toward the commercial antibody intact. Using two different proteins as models, we show that the cyclization tag developed here can be used as a specific tag for the backbone-cyclized protein, thereby facilitating detection and purification.