The figures show:
FIG. 1. Experimental strategy for transposon targeting using transposase-fusion proteins. The components of the targeting system include a transposable element that minimally contains the terminal inverted repeats containing the transposase binding sites (arrowheads), and may contain a gene of interest equipped with a suitable promoter. Targeting is achieved by fusing a targeting domain with the transposase. For transposition to occur, the fusion must not interfere with the activity of the transposase. (A) a fusion protein in which a specific DNA-targeting protein domain, responsible for binding to the target DNA, is fused to the transposase, thereby rendering a novel, and sequence-specific DNA-targeting function to it; (13) a fusion protein in which a protein domain interacts with an endogenous or engineered DNA-targeting protein; (C) a fusion protein in which a nucleolar localization signal directs the transposition complex into the nucleolus, which is composed of repetitive ribosomal RNA genes.
FIG. 2. A fusion protein consisting of the SB transposase and the tetracycline repressor retains functionality of both partners. (A) Schematic representation of the fusion protein that consists of the tetracycline repressor (TetR), a glycine-bridge, and the SB transposase. (B) Numbers of GFP-positive cells in the presence and absence of TetR/SB. A cell line containing the TRE-GFP reporter was transfected with the tetracycline transactivator and either beta-galactosidase or TetR/SB. Presence of TetR/SB decreases the number of GFP-positive cells, possibly by competing with the transactivator in binding to the TRE promoter region. (C). The TetR/SB fusion protein is active in transposition. HeLa cells were transfected with the T/neo transposon marker and either SB transposase or TetR/SB. G418-resistant colonies were counted.
The examples illustrate the invention.
REFERENCE EXAMPLE 1
Tagging The SB Transposase With Histidine-Tags
Histidine-tags were fused N-terminally and C-terminally to the Sleeping Beauty transposase by recombinant means. An N-terminal fusion completely abolished transposition activity, whereas a C-terminal tag reduced transposition activity to about 5-10% in vivo. Apparently, the SB transposase did not tolerate these additions, possibly due to an effect on protein folding. The N-terminal region of SB transposase contains two helix-turn-helix (HTH) domains responsible for specific binding of the transposase to the transposon inverted repeats. The function of the C-terminus is unknown, but this region of the protein is predicted to have a helical structure. C-terminal protein association determinants are present in different recombinases. For example, the crystal structure of Tn5 transposase, which acts as a dimer, shows that the main dimerization surface is provided by the C-terminus. The C-terminal regions of retroviral integrases were also found to encode multimerization functions. Taken together, it appears that protein tags interfere with transposition by compromising certain functions of the transposase, including DNA-binding and dimerization.
EXAMPLE 1
Implementation Of System Using Chimeric SB Transposase
In order to test the potential functionality of transposase fusion proteins in terms of both transposition and sequence-specific DNA-binding conferred by the fusion partner, a fusion protein was engineered containing the tetracycline repressor, a glycine-bridge and the Sleeping Beauty transposase (FIG. 2A, TetR/SB). The glycine-bridge consists of ten consecutive glycine residues, and its function is to form a flexible linker between the two functional folding units. Two different tests were undertaken: one that measures the ability of the fusion protein to bind tetracycline operator sequences, and the other that measures the ability of the fusion protein to catalyze transposition.
A human cell line derived from HeLa cells that contains a stably integrated GFP gene under the regulation of the tetracycline response element (TRE, encompassing seven units of the tetracycline operator) was transfected with a plasmid expressing the tetracycline transactivator in the absence or presence of TetR/SB, and the numbers of GFP-expressing cells were counted (FIG. 2B). The presence of TetR/SB drastically reduced the number of GFP-positive cells, indicating that it interferes with promoter activation at the TRE region, likely because it inhibits binding of the tetracycline transactivator protein to the operators.
Next, the ability of Tet/SB to catalyze transposition was examined in HeLa cells transfected with a transposon donor plasmid (T/neo, allowing selection with the antibiotic G418 after transposition) and either the SB transposase (positive control) or beta-galactosidase (negative control) or TetR/SB (FIG. 2C). After antibiotic selection for transformant cells, colonies were counted. The SB transposase increases the number of resistant cells about 30-50-fold, due to active transposition of the neo marker from plasmids to the chromosomes of cells. The TetR/SB fusion increased the number of resistant colonies about 2-2,5-fold. This result indicates that the fusion protein is active in transposition, but its activity is lower than that of the SB transposase.
Taken together, the results show that it is possible to generate fusion proteins containing the SB transposase and other sequence-specific DNA-binding proteins that retain the functionality of both partners.
Patent Application
20080008687
