Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells.
BACKGROUND
Cell-permeant Cre DNA site-specific recombinases provide an easily controlled means to regulate gene structure and function in living cells. Since recombination provides a stable and unambiguous record of protein uptake, the enzyme may also be used for quantitative studies of cis- and trans-acting factors that influence the delivery of proteins into cells.


RESULTS
In the present study, 11 recombinant fusion proteins were analyzed to characterize sequences and conditions that affect protein uptake and/or activity and to develop more active cell-permeant enzymes. We report that the native enzyme has a low, but intrinsic ability to enter cells. The most active Cre proteins tested contained either an N-terminal 6xHis tag and a nuclear localization sequence from SV40 large T antigen (HNC) or the HIV Tat transduction sequence and a C-terminal 6xHis tag (TCH6). The NLS and 6xHis elements separately enhanced the delivery of the HNC protein into cells; moreover, transduction sequences from fibroblast growth factor 4, HIV Tat or consisting of the (KFF)3K sequence were not required for efficient protein transduction and adversely affected enzyme solubility. Transduction of the HNC protein required 10 to 15 min for half-maximum uptake, was greatly decreased at 4 degrees C and was inhibited by serum. Efficient recombination was observed in all cell types tested (a T-cell line, NIH3T3, Cos7, murine ES cells, and primary splenocytes), and did not require localization of the enzyme to the nucleus.


CONCLUSIONS
The effects of different sequences on the delivery and/or activity of Cre in cultured cells could not be predicted in advance. Consequently, the process of developing more active cell-permeant recombinases was largely empirical. The HNC protein, with an excellent combination of activity, solubility and yield, will enhance the use of cell-permeant Cre proteins to regulate gene structure and function in living cells.
Lin
Qing
Q
Department of Microbiology and Immunology, Room AA5206, MCN, Vanderbilt University, School of Medicine, 1161 21st Ave South, AA4210, Nashville, TN 37232-2363, USA. qing.lin@vanderbilt.edu <qing.lin@vanderbilt.edu>
Jo
Daewoong
D
Gebre-Amlak
Kassatihun D
KD
Ruley
H Earl
HE
D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003470:Culture Media; D006639:Histidine / Q000737:chemistry; D019426:Integrases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D019913:Nuclear Localization Signals; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D011995:Recombination, Genetic; D013696:Temperature; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D003470:Culture Media; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C033223:polyhistidine; D006639:Histidine; C045073:Cre recombinase; D019426:Integrases