Cellular synthesis of non-linear proteins

Abstract

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 Nand C-termini, resp., of an elastin-like-protein (ELP), highly efficient cyclization of the ELP was obsd. At low protein synthesis rates (e.g., at low temp. in relatively poor media), the product was almost exclusively monomeric circular ELP; at high protein synthesis rates (e.g., at high temp. in rich media), the major product was monomeric circular protein while significant amts. of chain-extended oligomers were also obsd. The circular topol. has been demonstrated by SDS-PAGE, MALDI-TOF mass spectrometry, proteolytic digestion, and single-site mutation.