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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Main Authors: Ruley H Earl, Gebre-Amlak Kassatihun D, Jo Daewoong, Lin Qing
Format: Article
Language:English
Published: BMC 2004-10-01
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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AT jodaewoong enhancedcellpermeantcreproteinforsitespecificrecombinationinculturedcells
AT linqing enhancedcellpermeantcreproteinforsitespecificrecombinationinculturedcells