Protein trans-splicing on an M13 bacteriophage: towards directed evolution of a semisynthetic split intein by phage display.
Split inteins link their fused peptide or protein sequences with a peptide bond in an autocatalytic reaction called protein trans-splicing. This reaction is becoming increasingly important for a variety of applications in protein semisynthesis, polypeptide circularisation, construction of biosensors, or segmental isotopic labelling of proteins. However, split inteins exhibit greatly varying solubility, efficiency and tolerance towards the nature of the fused sequences as well as reaction conditions. We envisioned that phage display as an in vitro selection technique would provide a powerful tool for the directed evolution of split inteins with improved properties. As a first step towards this goal, we show that presentation of active split inteins on an M13 bacteriophage is feasible. Two different C-terminal intein fragments of the Ssp DnaB intein, artificially split at amino acid positions 104 and 11, were encoded in a phagemid vector in fusion to a truncated gpIII protein. For efficient production of hybrid phages, the presence of a soluble domain tag at their N-termini was necessary. Immunoblot analysis revealed that the hybrid phages supported protein trans-splicing with a protein or a synthetic peptide, respectively, containing the complementary intein fragment. Incorporation of biotin or desthiobiotin by this reaction provides a straightforward strategy for future enrichment of desired mutants from randomised libraries of the C-terminal intein fragments on streptavidin beads. Protein semisynthesis on a phage could also be exploited for the selection of chemically modified proteins with unique properties.
Garbe
Daniel
D
Fakultät Chemie-Chemische Biologie, Technische Universität Dortmund, Dortmund, Germany.
Thiel
Ilka V
IV
Mootz
Henning D
HD
D017104:Bacteriophage M13 / Q000235:genetics*; D019020:Directed Molecular Evolution; D047668:Inteins / Q000235:genetics*; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D019154:Protein Splicing; D020040:Trans-Splicing
D019151:Peptide Library; D010455:Peptides