Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells
<p>Abstract</p> <p>Background</p> <p>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 m...
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BMC
2004-10-01
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Series: | BMC Biotechnology |
Online Access: | http://www.biomedcentral.com/1472-6750/4/25 |
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author | Ruley H Earl Gebre-Amlak Kassatihun D Jo Daewoong Lin Qing |
author_facet | Ruley H Earl Gebre-Amlak Kassatihun D Jo Daewoong Lin Qing |
author_sort | Ruley H Earl |
collection | DOAJ |
description | <p>Abstract</p> <p>Background</p> <p>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 <it>cis</it>- and <it>trans</it>-acting factors that influence the delivery of proteins into cells.</p> <p>Results</p> <p>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 (TCH<sub>6</sub>). 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)<sub>3</sub>K 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°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.</p> <p>Conclusions</p> <p>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.</p> |
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spelling | doaj.art-6ada23a5d26640b38cbf2560aa1c0a542022-12-22T03:00:19ZengBMCBMC Biotechnology1472-67502004-10-01412510.1186/1472-6750-4-25Enhanced cell-permeant Cre protein for site-specific recombination in cultured cellsRuley H EarlGebre-Amlak Kassatihun DJo DaewoongLin Qing<p>Abstract</p> <p>Background</p> <p>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 <it>cis</it>- and <it>trans</it>-acting factors that influence the delivery of proteins into cells.</p> <p>Results</p> <p>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 (TCH<sub>6</sub>). 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)<sub>3</sub>K 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°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.</p> <p>Conclusions</p> <p>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.</p>http://www.biomedcentral.com/1472-6750/4/25 |
spellingShingle | Ruley H Earl Gebre-Amlak Kassatihun D Jo Daewoong Lin Qing Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells BMC Biotechnology |
title | Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells |
title_full | Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells |
title_fullStr | Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells |
title_full_unstemmed | Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells |
title_short | Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells |
title_sort | enhanced cell permeant cre protein for site specific recombination in cultured cells |
url | http://www.biomedcentral.com/1472-6750/4/25 |
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