The spontaneous isopeptide bond formation between a peptide tag (SpyTag) and its protein partner (SpyCatcher) allows the synthesis of non-linear proteins in cellular hosts. Strategic placement of sequences encoding SpyTag and SpyCatcher within protein-coding genes programs the post-translational modification of the expressed proteins in situ, and enables the synthesis of a variety of unconventional protein topologies. For example, when SpyTag and SpyCatcher were placed at the N- and C-termini, respectively, of an elastin-like-protein (ELP), highly efficient cyclization of the ELP was observed. At low protein synthesis rates (e.g., at low temperature in relatively poor media), the product was almost exclusively monomeric circular ELP; at high protein synthesis rates (e.g., at high temperature in rich media), the major product was monomeric circular protein while significant amounts of chain-extended oligomers were also observed. The circular topology has been demonstrated by SDS-PAGE, MALDI-TOF mass spectrometry, proteolytic digestion, and single-site mutation.

Cellular synthesis of non-linear proteins

246th National Meeting (2013)

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