title: Controlling macromolecular topology with genetically encoded SpyTag-SpyCatcher chemistry.
abstract: Control of molecular topology constitutes a fundamental challenge in macromolecular chemistry. Here we describe the synthesis and characterization of artificial elastin-like proteins (ELPs) with unconventional nonlinear topologies including circular, tadpole, star, and H-shaped proteins using genetically encoded SpyTag-SpyCatcher chemistry. SpyTag is a short polypeptide that binds its protein partner SpyCatcher and forms isopeptide bonds under physiological conditions. Sequences encoding SpyTag and SpyCatcher can be strategically placed into ELP genes to direct post-translational topological modification in situ. Placement of SpyTag at the N-terminus and SpyCatcher at the C-terminus directs formation of circular ELPs. Induction of expression at 16 °C with 10 μM IPTG yields 80% monomeric cyclic protein. When SpyTag is placed in the middle of the chain, it exhibits an even stronger tendency toward cyclization, yielding up to 94% monomeric tadpole proteins. Telechelic ELPs containing either SpyTag or SpyCatcher can be expressed, purified, and then coupled spontaneously upon mixing in vitro. Block proteins, 3-arm or 4-arm star proteins, and H-shaped proteins have been prepared, with the folded CnaB2 domain that results from the SpyTag-SpyCatcher reaction as the molecular core or branch junction. The modular character of the SpyTag-SpyCatcher strategy should make it useful for preparing nonlinear macromolecules of diverse sequence and structure.
authors: Division of Chemistry and Chemical Engineering, California Institute of Technology , Pasadena, California 91125, United States.
authors: Wen-Bin
authors: WB
authors: Zhang
authors: Fei
authors: F
authors: Sun
authors: David A
authors: DA
authors: Tirrell
authors: Frances H
authors: FH
authors: Arnold
mesh_terms: D000595:Amino Acid Sequence; D003500:Cyclization; D004549:Elastin / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007536:Isomerism; D047028:Macrocyclic Compounds / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D011506:Proteins / Q000737:chemistry* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics*
chemical_list: D047028:Macrocyclic Compounds; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D004549:Elastin