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

Abstract

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.Peer ReviewedPostprint (published version

Engineering Secretory Amyloids for Remote and Highly Selective Destruction of Metastatic Foci

“…Being insufficient to generate inflammation at the injection site (Figure C), this is still an issue to be considered during further IB development. However, since functional IBs or IB‐like particles have been successfully produced in endotoxin‐free strains of E. coli , in food‐grade lactic acid bacteria, and also in yeasts, the future clinically oriented development of secretory amyloids must necessarily consider these safer cell factories, favorable for the production of protein‐based drugs from strategic and regulatory point of views. Considering that any IB‐forming polypeptide with therapeutic value can also be engineered to acquire specific cell targeting, as in the case of T22‐PE24‐H6, the implantable material can be designed for precision medicines such as in cancer treatments.…”

Bacterial Inclusion Bodies: Discovering Their Better Half

“…Besides, the presence of functional aggregates had also been reported in the yeast species Schizosaccharomyces pombe [86] and Saccharomyces cerevisiae [87]. The controlled production of functional IB analogues in Pichia pastoris [88] and in the food-grade bacterium Lactococcus lactis has just been demonstrated [89]. Together with the recent description of IB fabrication in endotoxin-free E. coli [90,91], the production of IBs in microorganisms other than Gram-negative, endotoxin-containing bacteria is expanding the catalogues of cell factories for biologically safer IB-related products, what would conduct to a smoother implementation of IB-based protein delivery technologies in different fields.…”

Improving Biomaterials Imaging for Nanotechnology: Rapid Methods for Protein Localization at Ultrastructural Level

“…The green fluorescent protein (GFP) and three proteins of bovine ( Bos taurus sp.) origin, the catalytic domain of matrix metalloproteinase 2 (MMP‐2; from Tyr110 to Asp45 NM_174745) and 9 (MMP‐9; from Phe107 to Pro449 NM_174744), and the mature form of the interferon gamma (IFN‐γ; from Gln23 to Thr101 NM_173925) were used, as described in Ref …”

“…Ultrathin sections (70 nm) of selected areas of semithin sections (1 μm) of each sample were obtained with Leica ultracut UC6 microtome (Leica Microsystems), placed on carbon‐coated gold grids (200 mesh) and labeled in parallel for the four antigens of interest . Briefly, sections were blocked in 1% (w/v) bovine serum albumin (BSA; Sigma–Aldrich) in phosphate buffered saline (BSA/PBS) containing 20 mM of glycine (Sigma–Aldrich), incubated with the respective primary polyclonal primary antibody anti‐GFP (#ab6556, Abcam), anti‐MMP‐2 (#AV20016, Sigma–Aldrich), anti‐MMP‐9 (#50560‐RP01, Sino Biological Inc.), or anti‐IFN‐γ (#ab9657, Abcam) at working dilution 1:25, 1:5, 1:5, and 1:2, respectively, washed in BSA/PBS, incubated in protein A coupled to 10 nm‐gold particles (BBI Solutions) at working dilution 1:50, and washed firstly in PBS and then in deionized (MilliQ) water.…”