High-Efficiency Expression and Purification of DNAJB6b Based on the pH-Modulation of Solubility and Denaturant-Modulation of Size

The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual effects on kinetics and equilibrium are related to the unusually high chemical potential of DNAJB6b in solution. As a consequence, the chaperone alone forms highly polydisperse oligomers, whereas in a mixture with an amyloid-forming protein or peptide it may form co-aggregates to gain a reduced chemical potential, thus enabling the amyloid peptide to increase its chemical potential leading to enhanced solubility of the peptide. Understanding such action at the level of molecular driving forces and detailed structures requires access to highly pure and sequence homogeneous DNAJB6b with no sequence extension. We therefore outline here an expression and purification protocol of the protein “as is” with no tags leading to very high levels of pure protein based on its physicochemical properties, including size and charge. The versatility of the protocol is demonstrated through the expression of an isotope labelled protein and seven variants, and the purification of three of these. The activity of the protein is bench-marked using aggregation assays. Two of the variants are used to produce a palette of fluorescent DNAJB6b labelled at an engineered N- or C-terminal cysteine.

Chlorin Endogenous to the North Pacific Brittle Star Ophiura sarsii for Photodynamic Therapy Applications in Breast Cancer and Glioblastoma Models

Photodynamic therapy (PDT) represents a powerful avenue for anticancer treatment. PDT relies on the use of photosensitizers—compounds accumulating in the tumor and converted from benign to cytotoxic upon targeted photoactivation. We here describe (3S,4S)-14-Ethyl-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (ETPA) as a major metabolite of the North Pacific brittle stars Ophiura sarsii. As a chlorin, ETPA efficiently produces singlet oxygen upon red-light photoactivation and exerts powerful sub-micromolar phototoxicity against a panel of cancer cell lines in vitro. In a mouse model of glioblastoma, intravenous ETPA injection combined with targeted red laser irradiation induced strong necrotic ablation of the brain tumor. Along with the straightforward ETPA purification protocol and abundance of O. sarsii, these studies pave the way for the development of ETPA as a novel natural product-based photodynamic therapeutic.

Characterization of an Insoluble and Soluble Form of Melanin Produced by Streptomyces cavourensis SV 21, a Sea Cucumber Associated Bacterium

Melanin is a widely distributed and striking dark-colored pigment produced by countless living organisms. Although a wide range of bioactivities have been recognized, there are still major constraints in using melanin for biotechnological applications such as its fragmentary known chemical structure and its insolubility in inorganic and organic solvents. In this study, a bacterial culture of Streptomyces cavourensis SV 21 produced two distinct forms of melanin: (1) a particulate, insoluble form as well as (2) a rarely observed water-soluble form. The here presented novel, acid-free purification protocol of purified particulate melanin (PPM) and purified dissolved melanin (PDM) represents the basis for an in-depth comparison of their physicochemical and biological properties, which were compared to the traditional acid-based precipitation of melanin (AM) and to a synthetic melanin standard (SM). Our data show that the differences in solubility between PDM and PPM in aqueous solutions may be a result of different adjoining cation species, since the soluble PDM polymer is largely composed of Mg2+ ions and the insoluble PPM is dominated by Ca2+ ions. Furthermore, AM shared most properties with SM, which is likely attributed to a similar, acid-based production protocol. The here presented gentler approach of purifying melanin facilitates a new perspective of an intact form of soluble and insoluble melanin that is less chemical altered and thus closer to its original biological form.

Biocatalytic Production of Aldehydes: Exploring the Potential of Lathyrus cicera Amine Oxidase

Aldehydes are a class of carbonyl compounds widely used as intermediates in the pharmaceutical, cosmetic and food industries. To date, there are few fully enzymatic methods for synthesizing these highly reactive chemicals. In the present work, we explore the biocatalytic potential of an amino oxidase extracted from the etiolated shoots of Lathyrus cicera for the synthesis of value-added aldehydes, starting from the corresponding primary amines. In this frame, we have developed a completely chromatography-free purification protocol based on crossflow ultrafiltration, which makes the production of this enzyme easily scalable. Furthermore, we determined the kinetic parameters of the amine oxidase toward 20 differently substituted aliphatic and aromatic primary amines, and we developed a biocatalytic process for their conversion into the corresponding aldehydes. The reaction occurs in aqueous media at neutral pH in the presence of catalase, which removes the hydrogen peroxide produced during the reaction itself, contributing to the recycling of oxygen. A high conversion (>95%) was achieved within 3 h for all the tested compounds.

Cytotoxicity of Frutalin on Distinct Cancer Cells Is Independent of Its Glycosylation

Frutalin is a plant lectin with beneficial immunobiological action, although the access to its active form is still restricted. Moreover, there is a knowledge gap on isoform activity and glycosylation impact on its bioactivity, and recombinant production protocols were seen as ineffective. Here, a simpler and faster production and purification protocol was developed, attaining a yield of purified frutalin 3.3-fold higher than that obtained previously. Hemagglutination assays confirmed that this frutalin isoform could not agglutinate rabbit erythrocytes, while maintaining the native tetrameric structure, as indicated by DLS analysis, and strong interaction with methyl-alpha-galactose, in fluorescence spectroscopy studies. The cytotoxicity of the recombinant frutalin isoform was shown in a broad panel of human cancer cells: colon (HCT116), melanoma (A375), triple-negative breast cancer (MDA-MB-231), and ovarian (IGROV-1). Treatment with 8.5–11.8 μM TrxFTL reduced proliferation of all cancer cells to half in 48 h. This anti-proliferative effect encompasses the p53 pathway since it was significantly reduced in p53-null colon cancer cells (HCT116 p53−/−; GI50 of 25.0 ± 3.0 μM), when compared to the isogenic p53-positive cells (HCT116 p53+/+; GI50 of 8.7 ± 1.8 μM; p < 0.002). This recombinantly produced frutalin isoform has relevant cytotoxic effect and its biological activity is not dependent on glycosylation. The developed E. coli production and purification protocol generates high yield of non-glycosylated frutalin isoform with potent cytotoxic activity, enabling the development of novel anticancer p53-targeting therapies.

Mycobacterium tuberculosis ferritin: a suitable workhorse protein for cryo-EM development

The use of cryo-EM continues to expand worldwide and calls for good-quality standard proteins with simple protocols for their production. Here, a straightforward expression and purification protocol is presented that provides an apoferritin, bacterioferritin B (BfrB), from Mycobacterium tuberculosis with high yield and purity. A 2.12 Å resolution cryo-EM structure of BfrB is reported, showing the typical cage-like oligomer constituting of 24 monomers related by 432 symmetry. However, it also contains a unique C-terminal extension (164–181), which loops into the cage region of the shell and provides extra stability to the protein. Part of this region was ambiguous in previous crystal structures but could be built within the cryo-EM map. These findings and this protocol could serve the growing cryo-EM community in characterizing and pushing the limits of their electron microscopes and workflows.

MS2 Virus-like Particle as an Efficient Drug Delivery Tool: Optimizing the Synthesis and Purification Protocol

Introducing bacteriophage MS2 virus-like particles (VLPs) as gene and drug delivery tools increases the demand for optimizing their production and purification procedure. PEG precipitation method is used efficiently to purify VLPs, while the effects of pH and different electrolytes on the stability, size, and homogeneity of purified MS2 VLPs, and the encapsulated RNA sequences remained to be elucidated.In this regard, a vector, capable of producing VLP with an shRNA packed inside was prepared. The resulting VLPs in different buffers/solutions were assessed for their size, polydispersity index, and ability to protect the enclosed shRNA. We report that among Tris, HEPES, and PBS, with or without NaNO3, and also NaNO3 alone in different pH and ionic concentrations, the 100mM NaNO3-Tris buffer with pH:8 can be used as a new and optimal MS2 VLP production buffer, capable of inhibiting the VLPs aggregation. These VLPs show a size range of 27-30nm and suitable homogeneity with minimum 12-month stability at 4◦C. Moreover, the resulting MS2 VLPs were highly efficient and stable for at least 48 hours in conditions similar to in vivo. These features of MS2 VLPs produced in the newly introduced buffer make them an appropriate candidate for therapeutic agents’ delivery.