The impact of cell factory quality control on material properties is a neglected but critical issue in the fabrication of protein biomaterials, which are unique in merging structure and function. The ...molecular chaperoning of protein conformational status is revealed here as a potent molecular instructor of the macroscopic properties of self‐assembling, cell‐targeted protein nanoparticles, including biodistribution upon in vivo administration.
Inclusion bodies are insoluble protein aggregates usually found in recombinant bacteria when they are forced to produce heterologous protein species. These particles are formed by polypeptides that ...cross-interact through sterospecific contacts and that are steadily deposited in either the cell's cytoplasm or the periplasm. An important fraction of eukaryotic proteins form inclusion bodies in bacteria, which has posed major problems in the development of the biotechnology industry. Over the last decade, the fine dissection of the quality control system in bacteria and the recognition of the amyloid-like architecture of inclusion bodies have provided dramatic insights on the dynamic biology of these aggregates. We discuss here the relevant aspects, in the interface between cell physiology and structural biology, which make inclusion bodies unique models for the study of protein aggregation, amyloid formation and prion biology in a physiologically relevant background.
Inclusion bodies (50–500 nm in diameter) produced in recombinant bacteria can be engineered to contain functional proteins with therapeutic potential. Upon exposure, these protein particles are ...efficiently internalized by mammalian cells and promote recovery from diverse stresses. Being fully biocompatible, inclusion bodies are a novel platform, as tailored nanopills, for sustained drug release in advanced cell therapies.
In the present study the use of extracellular vesicles (EVs) as vehicles for therapeutic enzymes in lysosomal storage disorders was explored. EVs were isolated from mammalian cells overexpressing ...alpha‐galactosidase A (GLA) or N‐sulfoglucosamine sulfohydrolase (SGSH) enzymes, defective in Fabry and Sanfilippo A diseases, respectively. Direct purification of EVs from cell supernatants was found to be a simple and efficient method to obtain highly active GLA and SGSH proteins, even after EV lyophilization. Likewise, EVs carrying GLA (EV‐GLA) were rapidly uptaken and reached the lysosomes in cellular models of Fabry disease, restoring lysosomal functionality much more efficiently than the recombinant enzyme in clinical use. In vivo, EVs were well tolerated and distributed among all main organs, including the brain. DiR‐labelled EVs were localized in brain parenchyma 1 h after intra‐arterial (internal carotid artery) or intravenous (tail vein) administrations. Moreover, a single intravenous administration of EV‐GLA was able to reduce globotriaosylceramide (Gb3) substrate levels in clinically relevant tissues, such kidneys and brain. Overall, our results demonstrate that EVs from cells overexpressing lysosomal enzymes act as natural protein delivery systems, improving the activity and the efficacy of the recombinant proteins and facilitating their access to organs neglected by conventional enzyme replacement therapies.
The preparation of biological samples for electron microscopy is material‐ and time‐consuming because it is often based on long protocols that also may produce artifacts. Protein labeling for ...transmission electron microscopy (TEM) is such an example, taking several days. However, for protein‐based nanotechnology, high resolution imaging techniques are unique and crucial tools for studying the spatial distribution of these molecules, either alone or as components of biomaterials. In this paper, we tested two new short methods of immunolocalization for TEM, and compared them with a standard protocol in qualitative and quantitative approaches by using four protein‐based nanoparticles. We reported a significant increase of labeling per area of nanoparticle in both new methodologies (H = 19.811; p < 0.001) with all the model antigens tested: GFP (H = 22.115; p < 0.001), MMP‐2 (H = 19.579; p < 0.001), MMP‐9 (H = 7.567; p < 0.023), and IFN‐γ (H = 62.110; p < 0.001). We also found that the most suitable protocol for labeling depends on the nanoparticle's tendency to aggregate. Moreover, the shorter methods reduce artifacts, time (by 30%), residues, and reagents hindering, losing, or altering antigens, and obtaining a significant increase of protein localization (of about 200%). Overall, this study makes a step forward in the development of optimized protocols for the nanoscale localization of peptides and proteins within new biomaterials.
High resolution imaging techniques are essential tools for studying the spatial distribution of protein‐based materials at nanoscale. However, the traditional long protocols are material‐ and time‐consuming. In this work, authors present two new short approaches of post‐embedding TEM immunolocalization, using four types of protein aggregates with nanomedicine use as models. This study provides two important advantages, namely a significant increase of labeling and a general time reduction of protocol.
Bacterial inclusion bodies are intriguing nanoparticulate materials produced by biological systems in inexpensive processes whose nanometer‐scale features can be finely tailored during biological ...production. This novel, mechanically stable, biocompatible nanomaterial is shown to be suitable for 2D cell growth engineering, among other potential biomedical applications.
Many enzymes or fluorescent proteins produced in Escherichia coli are enzymatically active or fluorescent respectively when deposited as inclusion bodies. The occurrence of insoluble but functional ...protein species with native-like secondary structure indicates that solubility and conformational quality of recombinant proteins are not coincident parameters, and suggests that both properties can be engineered independently. We have here proven this principle by producing elevated yields of a highly fluorescent but insoluble green fluorescent protein (GFP) in a DnaK⁻ background, and further enhancing its solubility through adjusting the growth temperature and GFP gene expression rate. The success of such a two-step approach confirms the independent control of solubility and conformational quality, advocates for new routes towards high quality protein production and intriguingly, proves that high protein yields dramatically compromise the conformational quality of soluble versions. Biotechnol. Bioeng. 2008;101: 1353-1358.
Many enzymes of industrial interest are not in the market since they are bio-produced as bacterial inclusion bodies, believed to be biologically inert aggregates of insoluble protein.
By using two ...structurally and functionally different model enzymes and two fluorescent proteins we show that physiological aggregation in bacteria might only result in a moderate loss of biological activity and that inclusion bodies can be used in reaction mixtures for efficient catalysis.
This observation offers promising possibilities for the exploration of inclusion bodies as catalysts for industrial purposes, without any previous protein-refolding step.
Lactic acid bacteria (LAB) have a long history of safe exploitation by humans, being used for centuries in food production and preservation and as probiotic agents to promote human health. ...Interestingly, some species of these Gram-positive bacteria, which are generally recognized as safe organisms by the US Food and Drug Administration (FDA), are able to survive through the gastrointestinal tract (GIT), being capable to reach and colonize the intestine, where they play an important role. Besides, during the last decades, an important effort has been done for the development of tools to use LAB as microbial cell factories for the production of proteins of interest. Given the need to develop effective strategies for the delivery of prophylactic and therapeutic molecules, LAB have appeared as an appealing option for the oral, intranasal and vaginal delivery of such molecules. So far, these genetically modified organisms have been successfully used as vehicles for delivering functional proteins to mucosal tissues in the treatment of many different pathologies including GIT related pathologies, diabetes, cancer and viral infections, among others. Interestingly, the administration of such microorganisms would suppose a significant decrease in the production cost of the treatments agents since being live organisms, such vectors would be able to autonomously amplify and produce and deliver the protein of interest. In this context, this review aims to provide an overview of the use of LAB engineered as a promising alternative as well as a safety delivery platform of recombinant proteins for the treatment of a wide range of diseases.
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