Controlling macromolecular topology with genetically encoded SpyTag-SpyCatcher chemistry

J Am Chem Soc. 2013 Sep 18;135(37):13988-97. doi: 10.1021/ja4076452. Epub 2013 Sep 9.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Cyclization
  • Elastin / chemistry
  • Elastin / genetics
  • Electrophoresis, Polyacrylamide Gel
  • Isomerism
  • Macrocyclic Compounds / chemistry*
  • Molecular Sequence Data
  • Peptides / chemistry
  • Peptides / genetics
  • Proteins / chemistry*
  • Proteins / genetics*
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics*

Substances

  • Macrocyclic Compounds
  • Peptides
  • Proteins
  • Recombinant Fusion Proteins
  • Elastin