Abstract
Many different DNA delivery vehicles have been developed and tested, all with their advantages and disadvantages. The bacteriophage phi29 terminal protein (TP) is covalently linked to the 5’ ends of the phage genome during the DNA replication process. Our approach is to utilize this TP as a platform to incorporate different protein or peptide modules that can target the DNA to the interior of the cell, to the nucleus, or even to subcellular compartments. In order to be able to insert different peptide modules on the TP sequence to endow it with desired functions and/or eliminate unwanted regions of the protein, we have carried out a transposition screening to detect insertion-permissive points on the sequence of the TP. We report the functional characterization of 12 insertion mutants of the TP, and the identification of one site at position 38 that allows the insertion of peptides up to 17 amino acids in length while maintaining the ability of the TP to support DNA amplification in vitro. A protein with one insertion at that position containing a cysteine residue, a linker, and a thrombin recognition site was purified and its amplification activity was optimized.
Highlights
DNA delivery into eukaryotic cells is a task that ranges from trivial to extremely complicated depending on the exact outcome intended
In a somewhat unexpected finding we have shown that the phi29 terminal protein (TP), when expressed in mammalian cells, efficiently localizes in the nucleus and we were able to show that TP amino acids 14 to 38 constitute a bona fide eukaryotic nuclear localization signal (NLS) [17]
We considered that the whole GFP protein could be too voluminous to allow the correct folding of the TP and be compatible with its function, and that, for our purposes, a smaller insertion could be functional and still useful as a probe for permissive sites
Summary
DNA delivery into eukaryotic cells is a task that ranges from trivial to extremely complicated depending on the exact outcome intended. In order to achieve in vivo efficiency, quantitative yield, or subcellular compartment targeting, a large number of approaches have been tested [1]. These delivery methods can be subdivided in two: viral vectors and non-viral protocols [2, 3]. The viral vectors enjoy high efficiency and in some cases can be targeted to specific cell types, the method is complex, expensive, and concerns about safety remain. Non-viral vectors, on the other hand, are easier to use but are much less efficient and the targeting is difficult. There is effectively no system that enjoys the advantages of the two methods while not being affected by their inconvenients
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