Open AccessPhage TP901-1 Site-Specific Integrase Functions in Human Cells
Author(s) -
Stephanie M Stoll,
Daniel S. Ginsburg,
Michèle P. Calos
Publication year - 2002
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.184.13.3657-3663.2002
Subject(s) - integrase , biology , plasmid , escherichia coli , lactococcus lactis , bacteriophage , integrases , recombinant dna , integrase inhibitor , microbiology and biotechnology , dna , biochemistry , bacteria , virology , gene , genetics , virus , lactic acid , antiretroviral therapy , viral load
We demonstrate that the site-specific integrase encoded by phage TP901-1 of Lactococcus lactis subsp. cremoris has potential as a tool for engineering mammalian genomes. We constructed vectors that express this integrase in Escherichia coli and in mammalian cells and developed a simple plasmid assay to measure the frequency of intramolecular integration mediated by the integrase. We used the assay to document that the integrase functions efficiently in E. coli and determined that for complete reaction in E. coli, the minimal sizes of attB and attP are 31 and 50 bp, respectively. We carried out partial purification of TP901-1 integrase protein and demonstrated its functional activity in vitro in the absence of added cofactors, characterizing the time course and temperature optimum of the reaction. Finally, we showed that when expressed in human cells, the TP901-1 integrase carries out efficient intramolecular integration on a transfected plasmid substrate in the human cell environment. The TP901-1 phage integrase thus represents a new reagent for manipulating DNA in living mammalian cells.