Functional protein-based nanomaterial produced in microorganisms recognized as safe: A new platform for biotechnology

Cano Garrido, Olivia et al.Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest.This work was supported by grants from INIA, MINECO, Spain to AA and EGF (RTA2012-00028-C02-02), from DGI to JV (BeWell CTQ2013-40480-R), from MINECO to EGF and IR (MAT2013-50036-EXP) and to ICMAB (Severo Ochoa Programme for Centres of Excellence in R&D – SEV – 2015-0496), from Agència de Gestió d’Ajuts Universitaris i de Recerca to AV and JV (2014SGR-132 and 2014-SGR-17, respectively) and from EU to JV, AV and IR (H2020-INFRAIA-2014-2015; NFFA-654360). Besides, the authors acknowledge the financial support granted to AV and JV from the Centro de Investigación Biomédica en Red (CIBER) de Bioingeniería, Biomateriales y Nanomedicina financed by the Instituto de Salud Carlos III with assistance from the European Regional Development. OCG and WIT received a PhD fellowship from MECD (FPU) and CSIC (JAE-pre), respectively, and EGF a post-doctoral fellowship from INIA (DOC-INIA). AV has been distinguished with an ICREA ACADEMIA Award. The authors also acknowledge Micalis Institute, INRA, France that kindly provide us the strain clpP− htrA−-NZ9000 (patent n° EP1141337B1/US6994997B1). We are also indebted to “Servei de Microscopia” (UAB) and ICTS “NANBIOSIS”, more specifically to the Protein Production Platform of CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN)/IBB, at the UAB SepBioES scientific-technical service (http://www.nanbiosis.es/unit/u1-protein-production-platform-ppp/). No other potential conflicts of interest were identified.Peer reviewe