Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells

Qing Lin,Kassatihun D Grebre-Amlak,H Earl Ruley,Daewoong Jo

doi:10.1186/1472-6750-4-25

Abstract

BackgroundCell-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.ResultsIn 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°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.ConclusionsThe 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.

Highlights

Cell-permeant Cre DNA site-specific recombinases provide an controlled means to regulate gene structure and function in living cells
HT7N'C is similar to HNC except the His tag contains 11 additional amino acids (MASMTGGQQMG) from the pET28a(+) polylinker and the arginine of the nuclear localization sequence (NLS) sequence was converted to a lysine
HNCM, described previously [8], contains a membrane translocation sequence (MTS) from the leader sequence of fibroblast growth factor 4 (FGF-4) positioned at the C-terminus of HNC

Summary

Introduction

Cell-permeant Cre DNA site-specific recombinases provide an controlled means to regulate gene structure and function in living cells. The use of Cre expression vectors is constrained by the fact that prolonged exposure to the enzyme can be lethal to cells [4,6,7] To address these problems, we [8] and others [9,10,11,12] have developed membrane-permeable Cre recombinase proteins that are capable of entering cells by a process of protein transduction. Cell-permeant Cre proteins provide an effective means to regulate gene structure and function in living cells, and Cre-mediated recombination provides a potentially useful reporter system with which to study the process of protein transduction itself. Recombination provides a stable and quantitative record of protein uptake that circumvents problems of distinguishing between internalized and cell-associated proteins [16]

Methods

Results

Discussion

Conclusion