Abstract
BackgroundCell-penetrating peptides (CPPs) have been widely used as carriers to transport different molecules into living cells, whereas messenger RNAs (mRNAs) have been utilized as target molecules for the prevention and treatment of various diseases. However, the instability of CPPs and mRNAs has limited their application. Bacteriophage PP7 virus-like particles (VLPs) may protect peptides and RNAs from degradation through displaying foreign peptides on their surface and encapsidating RNA linked with the pac site.ResultsIn this study, the cDNA of the PP7 coat protein single-chain dimer carrying low molecular weight protamine (LMWP) and the cDNA of green fluorescent protein (GFP) were inserted into two multiple cloning sites of pETDuet-1, respectively. PP7 VLPs carrying the LMWP peptide and GFP mRNA were subsequently expressed in Escherichia coli BL21 (DE3) with high yield and thermal stability, and were easily purified. The VLPs were also non-replicative, non-infectious, and non-toxic. Moreover, they penetrated the mouse prostate cancer cells RM-1 after 24 h incubation. Last, PP7 VLPs carrying the LMWP could encapsidate the GFP mRNA, which was translated into mature protein in mammalian cells.ConclusionsRecombinant PP7 VLPs can be used simultaneously as a targeted delivery vector for both peptides and mRNA due to their abilities to package RNA and display peptides.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0274-9) contains supplementary material, which is available to authorized users.
Highlights
Cell-penetrating peptides (CPPs) have been widely used as carriers to transport different molecules into living cells, whereas messenger RNAs have been utilized as target molecules for the prevention and treatment of various diseases
The western blotting results showed the virus-like particle (VLP) existed in the first elution peak (Figs. 2b and 3a), and the expression levels of the purified 2PP7 VLPs, 2PP7-Protamine VLPs, and 2PP7-Protamine-green fluorescent protein (GFP) VLPs extracted from the supernatant of 1 L of overnight culture after sonication were approximately 5.56, 2.42, and 1.45 mg/L, respectively. These results demonstrated that PP7 VLPs carrying low molecular weight protamine (LMWP) or not carrying LMWP could be expressed in E. coli BL21 (DE3) with high-efficiency; the fusion of the peptide to PP7 coat protein and the inserted GFP gene in the second multiple cloning sites (MCSs) of the plasmid pETDuet-2PP7-Protamine decreased the expression of the PP7 VLPs
The higher level of GFP messenger RNA (mRNA) was observed in thecells incubated with 2PP7-Protamine-GFP VLPs (P < 0.001). In this group, the level of GFP mRNA at 48 h was significantly higher than that at 24 h. All these results showed that PP7 VLPs carrying the LMWP peptide could penetrate cell membrane and that the mRNA packaged by them was translated into mature protein in mammalian cells
Summary
Cell-penetrating peptides (CPPs) have been widely used as carriers to transport different molecules into living cells, whereas messenger RNAs (mRNAs) have been utilized as target molecules for the prevention and treatment of various diseases. Cell-penetrating peptides (CPPs) are short positively charged peptides containing less than 30 amino acid residues They exhibit low toxicity and have the capacity to cross cellular membranes by energy-dependent and/or independent mechanisms [1, 2]. CPPs can transport different molecules such as nanoparticles [3], liposomes [4], proteins [5], peptides [5], and oligonucleotides [5,6,7] into living cells Different delivery strategies such as direct conjugation, coincubation, and non-covalent or covalent cross-linking of CPPs with target molecules [4,5,6,7,8,9] have been investigated so far.
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