Macrocyclization
has been touted as an effective strategy to enhance
the in vivo stability and efficacy of protein therapeutics. Herein,
we describe a scalable and robust system based on the endogenous biosynthesis
of a noncanonical amino acid coupled to the pyrrolysine translational
machinery for the generation of lasso-grafted proteins. Thein cellulobiosynthesis of the noncanonical amino acidd-Cys-ε-Lys was achieved by hijacking the pyrrolysine
biosynthesis pathway, and then, its genetical incorporation into proteins
was performed using an optimized PylRS/tRNAPylpair and
cell line. This system was then applied to the structurally inspired
cyclization of a 23-mer therapeutic P16 peptide engrafted on a fusion
protein, resulting in near-complete cyclization of the target cyclic
subunit in under 3 h. The resulting cyclic P16 peptide fusion protein
possessed much higher CDK4 binding affinity than its linear counterpart.
Furthermore, a bifunctional bicyclic protein harboring a cyclic cancer
cell targeting RGD motif on the one end and the cyclic P16 peptide
on the other is produced and shown to be a potent cell cycle arrestor
with improved serum stability.