Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells

Abstract

BackgroundWe present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments.ResultsK14 IBs were electroporated into SW13 cells grown in culture together with a “reporter” plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5.ConclusionsSoluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells.

Production of functional inclusion bodies in endotoxin-free Escherichia coli

“…this fact could be of bionanotechnological relevance if in this strain, the insoluble protein is organized as inclusion bodies (IBs). IBs, as sub-micron protein particles, are gaining relevance in biotechnology ) and biomedicine ) because of their penetrability in mammalian cells in absence of toxicity and their ability to release therapeutic proteins inside receiving cells, in either substitutive protein therapies (Liovic et al 2012;Talafova et al 2013;Vazquez et al 2012) or in tissue engineering Cano-Garrido et al 2013;Seras-Franzoso et al 2013b;Seras-Franzoso et al 2013a). Interestingly, they act as natural biomimetics of hormone-releasing secretory granules of the endocrine system (Villaverde 2012), what offers a plethora of therapeutic opportunities.…”

“…However, we wanted to ensure that these particles still retained their ability to (i) mechanically stimulate the growth of mammalian cells when used as surface-decorating topographies in cell culture settings as described previously (García-Fruitós et al 2009;Seras-Franzoso et al 2013c;Diez-Gil et al 2010;Tatkiewicz et al 2013;Seras-Franzoso et al 2013b), and (ii) release functional proteins when internalized by mammalian cells (Liovic et al 2012;Vazquez et al 2012;Seras-Franzoso J et al 2013;Seras-Franzoso et al 2013b). The comparative analysis of cell proliferation on IB-decorated surfaces revealed similar properties of all tested IBs (Figure 7).…”

“…Due to their mechanical stability, the ability to penetrate mammalian cells in the absence of cellular damage and the release of functional protein, in a way similar to the release of functional hormones from amyloid repositories (Villaverde 2012), bacterial IBs became unexpectedly promising materials in drug delivery and in regenerative medicine Liovic et al 2012;Talafova et al 2013;García-Fruitós et al 2009;Seras-Franzoso et al 2012;Seras-Franzoso J et al 2013;Seras-Franzoso et al 2013c;Seras-Franzoso et al 2013b;Seras-Franzoso et al 2013a;Tatkiewicz et al 2013;Vazquez et al 2012;Villaverde et al 2012;Villaverde 2012). Although not determined quantitatively, contamination of IBs with bacterial LPS is a result of cell debris formation during cell disruption and separation (Neubauer et al 2006;Georgiou and Valax 1999).…”

“…In the context of potential applications of IBs in drug delivery either in vitro for regenerative medicine , in vivo in oral administration or envisaging transdermal or cell-targeted delivery (Talafova et al 2013;Liovic et al 2012), endotoxin-free IBs hold promise for the development towards a powerful tool that ensures both functionality and additional biosafety, being particularly attractive regarding cost-effective production and regulatory issues.…”

“…Bacterial IBs show high biological activity (Garcia-Fruitos et al 2005;GonzalezMontalban et al 2007;Garcia-Fruitos et al 2012), spontaneous penetrability in mammalian cells ) and also mechanical and biological stability García-Fruitós et al 2009), which has allowed to adapt them as nanostructured materials for surface decoration in tissue engineering (García-Fruitós et al 2009;Seras-Franzoso et al 2013b;Tatkiewicz et al 2013;Diez-Gil et al 2010;Seras-Franzoso et al 2011;Seras-Franzoso et al 2012;Seras-Franzoso J et al 2013) and in the context of top-down and bottom-up protein replacement/delivery cell therapies Cano-Garrido et al 2013;Talafova et al 2013;Liovic et al 2012). However, these applications require the bacterial product being free of E.…”