A Plasmid Display Platform for the Identification of New Functional Cell Penetrating Peptides

Cell Penetrating Peptides (CPPs) have received a great deal of attention since they have the ability to deliver various functional cargos to cells and tissues.  However, the use of CPPs in the clinic has been hampered by a lack of stability, specificity, and a full understanding of their mechanism of action.  Several groups have attempted to use combinatorial approaches to identify new CPPs from randomized peptide libraries, but the experiments have been challenging as the selection for peptides with a bona fide CPP phenotype is not trivial. 

Combinatorial methods in protein engineering are dependent on the ability to separate members of a library exhibiting a desirable phenotype. This is especially challenging with CPPs as the desired phenotype is the functional delivery of cargos to the cytosol of cells.  To address this problem we have chosen to work with an underused plasmid display (PD) technique^{1, 2}.  The plasmid is used to express a DNA binding domain, and we have fused CPPs to this protein (the p50 homodimer of NF-κB). When the protein is expressed, it provides a non-covalent attachment between the CPP and the plasmid DNA molecule which contains the CPP DNA sequence.  A unique feature of this approach as compared to other display techniques is that the plasmid DNA can also serve as the cargo to be delivered by the CPP.  We have included a fluorescent protein gene in the plasmid DNA so that expression of the fluorescent protein in the target cell unequivocally demonstrates that the CPP delivered the plasmid DNA to the cellular cytosol².  This novel PD platform enables the selection peptides with CPP phenotypes from a large combinatorial library of short peptides.

We have used this this platform for the selection of a randomized peptide library over PC12 cells, which are neuronal-like cells^{3, 4}.  This resulted in the identification of an unusual new CPP, called SG3, which is surprisingly not highly cationic like the majority of other known CPPs ⁵.  Fusion proteins with the SG3 peptide were expressed and purified, and the SG3 peptide is able to deliver the GFP protein to both PC12 cells and primary astrocytes, but on its own, it is not as efficient as the well known TAT CPP.  More interestingly, the PD protocol requires the addition of a cationic lipid during the selection procedure and when SG3 is combined with the lipid, it significantly outperforms the TAT peptide in both PC12 cells and cultured primary astrocytes.  To demonstrate the therapeutic potential of this new peptide, we explored synthetic peptides with CPPs attached to the pro-apoptotic BH3 peptide, and we were able to show that the BH3-SG3 with the cationic lipid significantly outperforms the BH3-TAT peptide in the killing of primary astrocytes, which was the desired outcome⁵.

This work demonstrates the power of the new PD-based selection platform for the identification of new CPPs.  And, the newly identified SG3 peptide is a new CPP that may be a valuable new tool for drug and gene delivery to cells.  By altering the PD selection system, it should be possible to select for CPPs with other desired traits such as cell and tissue type specificity.

1.         Gao, S., Simon, M.J., Morrison, B., 3rd & Banta, S. Bifunctional chimeric fusion proteins engineered for DNA delivery: optimization of the protein to DNA ratio. Biochim Biophys Acta 1790, 198-207 (2009).

2.         Gao, S., Simon, M.J., Morrison, B., 3rd & Banta, S. A Plasmid Display Platform for the Selection of Peptides Exhibiting a Functional Cell Penetrating Phenotype. Biotechnol Prog, (In Press) (2010).

3.         Simon, M.J., Gao, S., Kang, W.H., Banta, S. & Morrison, B., 3rd TAT-mediated intracellular protein delivery to primary brain cells is dependent on glycosaminoglycan expression. Biotechnol Bioeng 104, 10-19 (2009).

4.         Simon, M.J., Kang, W.H., Gao, S., Banta, S. & Morrison III, B. TAT Is Not Capable of Transcellular Delivery Across an Intact Endothelial Monolayer In Vitro. Ann Biomed Eng (2010).

5.         Gao, S., Simon, M.J., Morrison Iii, B. & Banta, S. An unusual cell penetrating peptide identified using a plasmid display-based functional selection platform. ACS Chem Biol, (Submitted, In Revision) (2010).