Probing the Biosafety of Implantable Artificial Secretory Granules for the Sustained Release of Bioactive Proteins.

Among bio-inspired protein materials, secretory protein microparticles are of clinical interest as self-contained, slow protein delivery platforms that mimic secretory granules of the human endocrine system, in which the protein is both the drug and the scaffold. Upon subcutaneous injection, their progressive disintegration results in the sustained release of the building block polypeptides, which reach the bloodstream for systemic distribution and subsequent biological effects. Such entities are easily fabricated in vitro by Zn-assisted cross-molecular coordination of histidine residues. Using cationic Zn for the assembly of selected pure protein species and in the absence of any heterologous holding material, these granules are expected to be nontoxic and therefore adequate for different clinical uses. However, such presumed biosafety has not been so far confirmed and the potential protein dosage threshold not probed yet. By selecting the receptor binding domain (RBD) from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein as a model protein and using a mouse lab model, we have explored the toxicity of RBD-made secretory granules at increasing doses up to ∼100 mg/kg of animal weight. By monitoring body weight and biochemical blood markers and through the histological scrutiny of main tissues and organs, we have not observed systemic toxicity. Otherwise, the bioavailability of the material was demonstrated by the induction of specific antibody responses. The presented data confirm the intrinsic biosafety of artificial secretory granules made by recombinant proteins and prompt their further clinical development as self-contained and dynamic protein reservoirs.

Recombinant Proteins for Assembling as Nano- and Micro-Scale Materials for Drug Delivery: A Host Comparative Overview.

By following simple protein engineering steps, recombinant proteins with promising applications in the field of drug delivery can be assembled in the form of functional materials of increasing complexity, either as nanoparticles or nanoparticle-leaking secretory microparticles. Among the suitable strategies for protein assembly, the use of histidine-rich tags in combination with coordinating divalent cations allows the construction of both categories of material out of pure polypeptide samples. Such molecular crosslinking results in chemically homogeneous protein particles with a defined composition, a fact that offers soft regulatory routes towards clinical applications for nanostructured protein-only drugs or for protein-based drug vehicles. Successes in the fabrication and final performance of these materials are expected, irrespective of the protein source. However, this fact has not yet been fully explored and confirmed. By taking the antigenic RBD domain of the SARS-CoV-2 spike glycoprotein as a model building block, we investigated the production of nanoparticles and secretory microparticles out of the versions of recombinant RBD produced by bacteria (Escherichia coli), insect cells (Sf9), and two different mammalian cell lines (namely HEK 293F and Expi293F). Although both functional nanoparticles and secretory microparticles were effectively generated in all cases, the technological and biological idiosyncrasy of each type of cell factory impacted the biophysical properties of the products. Therefore, the selection of a protein biofabrication platform is not irrelevant but instead is a significant factor in the upstream pipeline of protein assembly into supramolecular, complex, and functional materials.

VapD in Xylella fastidiosa Is a Thermostable Protein with Ribonuclease Activity.

Xylella fastidiosa strain 9a5c is a gram-negative phytopathogen that is the causal agent of citrus variegated chlorosis (CVC), a disease that is responsible for economic losses in Brazilian agriculture. The most well-known mechanism of pathogenicity for this bacterial pathogen is xylem vessel occlusion, which results from bacterial movement and the formation of biofilms. The molecular mechanisms underlying the virulence caused by biofilm formation are unknown. Here, we provide evidence showing that virulence-associated protein D in X. fastidiosa (Xf-VapD) is a thermostable protein with ribonuclease activity. Moreover, protein expression analyses in two X. fastidiosa strains, including virulent (Xf9a5c) and nonpathogenic (XfJ1a12) strains, showed that Xf-VapD was expressed during all phases of development in both strains and that increased expression was observed in Xf9a5c during biofilm growth. This study is an important step toward characterizing and improving our understanding of the biological significance of Xf-VapD and its potential functions in the CVC pathosystem.

A novel and enantioselective epoxide hydrolase from Aspergillus brasiliensis CCT 1435: purification and characterization.

A novel epoxide hydrolase from Aspergillus brasiliensis CCT1435 (AbEH) was cloned and overexpressed in Escherichia coli cells with a 6xHis-tag and purified by nickel affinity chromatography. Gel filtration analysis and circular dichroism measurements indicated that this novel AbEH is a homodimer in aqueous solution and contains the typical secondary structure of an α/ß hydrolase fold. The activity of AbEH was initially assessed using the fluorogenic probe O-(3,4-epoxybutyl) umbelliferone and was active in a broad range of pH (6-9) and temperature (25-45°C); showing optimum performance at pH 6.0 and 30°C. The Michaelis constant (KM) and maximum rate (Vmax) values were 495µM and 0.24µM/s, respectively. Racemic styrene oxide (SO) was used as a substrate to assess the AbEH activity and enantioselectivity, and 66% of the SO was hydrolyzed after only 5min of reaction, with the remaining (S)-SO ee exceeding 99% in a typical kinetic resolution behavior. The AbEH-catalyzed hydrolysis of SO was also evaluated in a biphasic system of water:isooctane; (R)-diol in 84% ee and unreacted (S)-SO in 36% ee were produced, with 43% conversion in 24h, indicating a discrete enantioconvergent behavior for AbEH. This novel epoxide hydrolase has biotechnological potential for the preparation of enantiopure epoxides or vicinal diols.

Initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa related to the human cytosolic 5'-nucleotidase I.

The 5'-nucleotidases constitute a ubiquitous family of enzymes that catalyze either the hydrolysis or the transfer of esterified phosphate at the 5' position of nucleoside monophosphates. These enzymes are responsible for the regulation of nucleotide and nucleoside levels in the cell and can interfere with the phosphorylation-dependent activation of nucleoside analogs used in therapies targeting solid tumors and viral infections. In the present study, we report the initial biochemical and functional characterization of a 5'-nucleotidase from Xylella fastidiosa that is related to the human cytosolic 5'-nucleotidase I. X. fastidiosa is a plant pathogenic bacterium that is responsible for numerous economically important crop diseases. Biochemical assays confirmed the phosphatase activity of the recombinant purified enzyme and revealed metal ion dependence for full enzyme activity. In addition, we investigated the involvement of Xf5'-Nt in the formation of X. fastidiosa biofilms, which are structures that occlude the xylem vessels of susceptible plants and are strictly associated with bacterial pathogenesis. Using polyclonal antibodies against Xf5'-Nt, we observed an overexpression of Xf5'-Nt during the initial phases of X. fastidiosa biofilm formation that was not observed during X. fastidiosa planktonic growth. Our results demonstrate that the de/phosphorylation network catalyzed by 5'-nucleotidases may play an important role in bacterial biofilm formation, thereby contributing novel insights into bacterial nucleotide metabolism and pathogenicity.

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coli.

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa.

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery.

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA:LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies.