Engineering Permissive Insertion Sites in the Bacteriophage Phi29 DNA-Linked Terminal Protein

PLoS One. 2016 Oct 25;11(10):e0164901. doi: 10.1371/journal.pone.0164901. eCollection 2016.


Many different DNA delivery vehicles have been developed and tested, all with their advantages and disadvantages. The bacteriophage phi29 terminal protein (TP) is covalently linked to the 5' ends of the phage genome during the DNA replication process. Our approach is to utilize this TP as a platform to incorporate different protein or peptide modules that can target the DNA to the interior of the cell, to the nucleus, or even to subcellular compartments. In order to be able to insert different peptide modules on the TP sequence to endow it with desired functions and/or eliminate unwanted regions of the protein, we have carried out a transposition screening to detect insertion-permissive points on the sequence of the TP. We report the functional characterization of 12 insertion mutants of the TP, and the identification of one site at position 38 that allows the insertion of peptides up to 17 amino acids in length while maintaining the ability of the TP to support DNA amplification in vitro. A protein with one insertion at that position containing a cysteine residue, a linker, and a thrombin recognition site was purified and its amplification activity was optimized.

MeSH terms

  • Bacteriophages / genetics*
  • Cysteine / genetics
  • DNA Replication
  • DNA Transposable Elements
  • DNA, Viral / genetics
  • Genetic Engineering / methods
  • Mutagenesis, Insertional / methods*
  • Peptides / genetics*
  • Peptides / metabolism
  • Viral Proteins / genetics*


  • DNA Transposable Elements
  • DNA, Viral
  • Peptides
  • Viral Proteins
  • Cysteine

Grant support

This work was supported by the Spanish Ministry of Economy and Competitiveness [BFU2014–52656 P to MS]; Fundación Ramón Areces institutional grant to the Centro de Biología Molecular ‘Severo Ochoa’. Predoctoral fellowship JAEdoc of Consejo Superior de Investigaciones Científicas to PG]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.