Non-amyloidogenic peptide tags for the regulatable self-assembling of protein-only nanoparticles
Introduction
Viral capsid proteins self-assemble as complex, highly symmetric particles that act as natural cages for the cell-targeted delivery of their genomes. The tailored construction of virus-inspired complexes is a promising route to drug delivery [1], [2], [3], [4], [5], [6], [7]. Being devoid of any infectious material, “artificial viruses” [8] do not show the undesired biological side effects associated to administration of viruses in viral gene therapy [9]. In this context, virus-like particles (VLPs), microbial organelles, [10], multifunctional proteins [11] and a spectrum of diverse vesicular materials are under development as carriers for therapeutic nucleic acids or conventional drugs. Many functional peptides have been identified from nature or selected by directed molecular evolution as ligands for cell surface receptors, membrane-active peptides and nuclear localization signals [4], [5], [6], [12]. When conveniently pooled, these tag-mediated activities confer virus-like properties to the resulting multifunctional entities. In protein-only vehicles, all these domains can be covalently combined in single chain molecules that constitute the monomeric building blocks [11]. However, peptides enabling their holding proteins to organize as nanosized particles have so far been unidentified. The so-called self-assembling peptides, that might have been potentially promising for nanoparticle generation, are in general amyloidogenic protein segments that form fibers, membranes or hydrogels [13], [14]. When used in fusion proteins, these peptides induce protein aggregation [15], [16], being useless as tags for nanoparticle formation. Therefore, promoting the assembling of a selected protein as nanoparticles is so far excluded from rational engineering.
We have very recently described that a nine-arginine peptide (R9), when displayed on the surface of a recombinant, His-tagged EGFP, promotes the self-assembling of the whole fusion protein as regular nanoparticles of about 20 nm in diameter [17]. These constructs efficiently penetrate cultured mammalian cells by an endocytic pathway [18], cross the nuclear membrane, accumulate in the nucleus and allow the expression of a carried transgene [17]. The formation of these supramolecular complexes is completely distinguishable from unspecific protein aggregation [7], [19], [20]. Cationic peptides, including poly-arginines of different lengths, are well known by their membrane-crossing and DNA-condensation abilities, and widely used in gene therapy and more generally in drug delivery [5], [6], [12], [21]. However, if showing a general applicability, such a newly described architectonic ability would be specially promising for the easy engineering of protein nanoparticles formed by specific proteins with desired biological activities.
To explore the possibility of effectively controlling the assembly of protein nanoparticles, we have examined here the role of cationic peptides and poly-histidines as an architectonic tag pair. These agents, upon incorporated into monomeric building blocks, synergistically cooperate in promoting nanoparticle formation by balancing, in a regulatable way, protein–protein and protein–DNA interactions. The potential of the ‘nano-architectonic tag’ concept is discussed here in the context of the design of smart, protein-based particles by conventional genetic engineering.
Section snippets
Protein design and gene cloning
Several derivatives of R9–GFP–H6 containing decreasing numbers of arginine residues were constructed in house by site directed mutagenesis of the parental clone, by replacing these residues by glycines and alanines to keep the length of the peptide tag constant (Table 1). The new constructs R7–GFP–H6, R6–GFP–H6 and R3–GFP–H6 were efficiently produced in Escherichia coli Rosetta from the vector pET21b (Novagen 69744-3). Nine additional derivatives of GFP–H6 containing diverse amino terminal
Mapping the architectonic abilities of poly-arginines
While His-tagged GFP is exclusively found in a disassembled monomeric form (of around 5 nm), the addition of the cell-penetrating poly-arginine (R9) peptide at the amino terminus promoted the spontaneous organization of R9–GFP–H6 as building blocks of regularly sized nanoparticles of around 20 nm [17]. To map the architectonic properties of poly-arginines we constructed a series of arginine-based tags (Rn) with a decreasing number of arginine residues, to evaluate if they retained the ability to
Discussion
The construction of self-assembling protein-only nanoparticles from repetitive building blocks has been a rather neglected issue in nanomedicine, in contrast to the long run expertise accumulated in the fabrication of liposomes and polymeric particles with pre-defined nanoscale features [35], [36], [37], [38], [39]. Consequently, the current protein-based vehicles for drug delivery generated de novo include a catalog of rather amorphous entities [40] that are produced under no previous design.
Conclusion
The combination of a cationic peptide and a hexa-histidine tail fused to the amino and carboxy termini, respectively, of different proteins enable them to act as building blocks of self-assembling nanoparticles whose properties are regulatable by pH during particle formation. These vehicles are also able to condense and deliver expressible DNA into mammalian cells. The architectonic properties of the tag pair at the nanoscale are supported by electrostatic contacts, primarily driven by the
Acknowledgments
We appreciate the technical support of Fran Cortés from the Cell Culture Unit of Servei de Cultius Cel·lulars, Producció d’Anticossos i Citometria (SCAC), and from Servei de Microscòpia, both at the UAB, and the Protein Production Platform (PPP) of the CIBER de Bioingeniería, Biomateriales y Nanomedicina. We also acknowledge the financial support received for the design and production of artificial viruses for gene therapy to EV and AV from FISS (PS0900165), MINECO (ACI2009-0919), AGAUR (
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