Expression, Purification and Biological Activity of a Cell-permeable Cre Recombinase

The Cre/lox P site-specific recombinase system, which has two components: Cre recombinase and two 34-bp lox P sites that Cre recognizes, has been widely used in conditional gene knockout/activation to study the structure and functions of gene. In the present study, a cell-permeable fusion protein (His6-NLS-Cre-MTS) containing a 12-amino acid membrane translocation sequence (MTS), a nuclear localization signal (NLS) and an N-terminal His6 affinity tag, was expressed in BL21 strains (e.g., DE3) transformed with pDJHisCre by induction of IPTG. The fusion protein was purified with His-Bond Ni-NTA resin. Its functionality was confirmed in a cell-free recombination assay with a plasmid (e.g., pApoE-SCS-EGFP) containing lox P-flanked gene(s), and in an intracellular recombination system using lox P-flanked STOP cassette-modified BEL-7402 cells, by assaying the expression of enhanced green fluorescent protein (EGFP). This cell-permeable Cre recombinase provides a rapid alternative means of manipulating mammalian gene structure and function in vitro and in vivo. Its advantages and potential uses are discussed.