A novel approach for the purification of aggregation prone proteins

The protein aggregation is one of the major challenges of the biotechnological industry, especially in the areas of development and commercialization of successful protein-based drug products. The inherent high aggregation tendency of proteins during various manufacturing processes, storage, and administration has significant impact upon the product quality, safety and efficacy. We have developed an interesting protein purification approach that separates the functionally active protein from inactive aggregates using a detergent concentration gradient. The C-terminally His tagged nucleocapsid protein of Crimean Congo Hemorrhagic fever virus (CCHFV) has high aggregation tendency and rapidly precipitates upon purification by NiNTA chromatography. Using the new purification approach reported here, the freshly purified protein by NiNTA chromatography was further processed using a detergent gradient. In this new purification approach the active protein is retained in the low detergent concentration zone while the inactive aggregates are promptly removed by their rapid migration to the high detergent concentration zone. The method prevented further aggregation and retained the RNA binding activity in the native protein despite numerous freeze thaw cycles. This simple approach prevents protein aggregation by rapidly separating the preformed early aggregates and creating the appropriate microenvironment for correctly folded proteins to retain their biological activity. It will be of potential importance to the biotechnological industry and other fields of protein biochemistry that routinely face the challenges of protein aggregation.

Spectroscopic Fourier Transform Infrared Study of Poly (N-Isopropylacrylamide)-Human Recombinant Platelet-Derived Growth Factor- BB Protein as a Strategic Biobetter Regenerative Material

Biobetter or Biosuperior are recombinant protein drugs with one or more characteristics that are better than the original formulation. Platelet-Derived Growth Factor (PDGF) is a potent mitogen that can induce pulp stem cell proliferation and promote angiogenesis in vitro. PDGF-BB is required for wound healing, and they are chemoattractants that can activate macrophages and fibroblast activation in vitro. Poly(n-isopropylacrylamide) (PNIPAM) polymer is a thermoresponsive polymer widely used for drug delivery. A lyophilized combination of PNIPAM and 5µg of Recombinant Human PDGF-BB(rhPDGF-BB)(sigma Aldrich-P3201) protein was prepared in the present study. This combination was subjected to Spectroscopic Fourier-Transform Infrared(FTIR) study. The results suggested that a biophysical approach like FTIR enables assessing protein stability and aggregation tendency of recombinant proteins, contributing as a useful method to identify samples with prospective high therapeutic values.

Spatial structure of Globularia bisnagarica L. (Plantaginaceae, Magnoliopsida) coenopopulations

The paper presents the results of the spatial distribution analysis of 18 Globularia bisnagarica L. coenopopulations in the Saratov, Samara, Ulyanovsk and Orenburg regions and the Republic of Tatarstan. The analysis was carried out with the Spatstat package of the R environment for statistical computing. The research reveals an aggregation tendency for the G. bisnagarica coenopopulations. It is suggested that this tendency may be attributed to the prevalence of seed reproduction and certain peculiarities of dissemination (barochory) in the studied coenopopulations as well as to the confinement of the species to eroded landforms (slopes of watersheds, gullies, runoff hollows, etc.) characterized by high heterogeneity of environmental conditions. It has been established that the majority of G. bisnagarica coenopopulations grow in heavily or moderately sodded habitats. Nevertheless, in the studied communities, G. bisnagarica occurs abundantly and dominates along with Stipa pennata, Bromus inermis, Salvia nutans, Elytrigia repens, Poa compressa, etc. Finally, the spatial structure of G. bisnagarica coenopopulations is impacted by the interspecific competition in calciphilous phytocenoses under the invasion of eurybiontic steppe grasses.

Behavioral Biomarker Responses of Filopaludina bengalensis to Acute Copper Toxicity

As heavy metals are bioaccumulative and toxic in nature, increasing heavy metal levels in the environment is of great concern. The present study was aimed to assess the acute toxicity effects of copper on some behavioral attributes of Filopaludina bengalensis (locally known as ‘Genri’), a freshwater gastropod, under laboratory condition. Exposure to copper (0.50-2.0 ppm) for 96 h resulted in significant mortality. Recorded LC50 value at 96h was 0.586 ppm. The aggregation tendency, crawling activity and mucus secretion were observed at different exposure period upon acute exposure of copper (0.586 ppm).With the advancement of exposure period, gradual deaggregation was observed in snails of copper treated sets. There was statistically significant difference (P <0.01) in aggregation response and foot protrusion response at different span of exposure in copper treated groups. In copper treated sets, a substantial increase (P<0.01) in mucus secretion (%) was evident with time. Foot protrusion and movement was inhibited in copper treated groups. All three responses in the copper treated groups were significantly different (P <0.01) from that of control groups. The behavioral responses observed in this study of Filopaludina bengalensis could be used as biomarkers for copper contamination in any aquatic ecosystems.

ELISA- and Activity Assay-Based Quantification of BMP-2 Released In Vitro Can Be Biased by Solubility in “Physiological” Buffers and an Interfering Effect of Chitosan

Chitosan nanogel-coated polycaprolactone (PCL) fiber mat-based implant prototypes with tailored release of bone morphogenic protein 2 (BMP-2) are a promising approach to achieve implant-mediated bone regeneration. In order to ensure reliable in vitro release results, the robustness of a commercially available ELISA for E. coli-derived BMP-2 and the parallel determination of BMP-2 recovery using a quantitative biological activity assay were investigated within a common release setup, with special reference to solubility and matrix effects. Without bovine serum albumin and Tween 20 as solubilizing additives to release media buffed at physiological pH, BMP‑2 recoveries after release were notably reduced. In contrast, the addition of chitosan to release samples caused an excessive recovery. A possible explanation for these effects is the reversible aggregation tendency of BMP-2, which might be influenced by an interaction with chitosan. The interfering effects highlighted in this study are of great importance for bio-assay-based BMP-2 quantification, especially in the context of pharmaceutical release experiments.

Modified Nanofibrous Filters with Durable Antibacterial Properties

The main aims of the research were to produce efficient nanofibrous filters with long-term antibacterial properties and to confirm the functionality of samples under real filtration conditions. A polyurethane solution was modified by micro- or nanoparticles of copper oxide in order to juxtapose the aggregation tendency of particles depending on their size. Modified solutions were electrospun by the Nanospider technique. The roller spinning electrode with a needle surface and static wire electrode were used for the production of functionalized nanofibers. The antibacterial properties of the modified nanofibrous layers were studied under simulated conditions of water and air filtration. Particular attention was paid to the fixation mechanism of modifiers in the structure of filters. It was determined that the rotating electrode with the needle surface is more efficient for the spinning of composite solutions due to the continuous mixing and the avoidance of particle precipitation at the bottom of the bath with modified polyurethane. Moreover, it was possible to state that microparticles of copper oxide are more appropriate antimicrobial additives due to their weaker aggregation tendency but stronger fixation in the fibrous structure than nanoparticles. From the results, it is possible to conclude that nanofibers with well-studied durable antibacterial properties may be recommended as excellent materials for water and air filtration applications.

Antibiotic Potential and Biophysical Characterization of Amphipathic β-Stranded [XZ]n Peptides With Alternating Cationic and Hydrophobic Residues

Cationic membrane-active peptides are considered to be promising candidates for antibiotic treatment. Many natural and artificial sequences show an antimicrobial activity when they are able to take on an amphipathic fold upon membrane binding, which in turn perturbs the integrity of the lipid bilayer. Most known structures are α-helices and β-hairpins, but also cyclic knots and other irregular conformations are known. Linear β-stranded antimicrobial peptides are not so common in nature, but numerous model sequences have been designed. Interestingly, many of them tend to be highly membranolytic, but also have a significant tendency to self-assemble into β-sheets by hydrogen-bonding. In this minireview we examine the literature on such amphipathic peptides consisting of simple repetitive sequences of alternating cationic and hydrophobic residues, and discuss their advantages and disadvantages. Their interactions with lipids have been characterized with a number of biophysical techniques—especially circular dichroism, fluorescence, and infrared—in order to determine their secondary structure, membrane binding, aggregation tendency, and ability to permeabilize vesicles. Their activities against bacteria, biofilms, erythrocytes, and human cells have also been studied using biological assays. In line with the main scope of this Special Issue, we attempt to correlate the biophysical results with the biological data, and in particular we discuss which properties (length, charge, aggregation tendency, etc.) of these simple model peptides are most relevant for their biological function. The overview presented here offers ideas for future experiments, and also suggests a few design rules for promising β-stranded peptides to develop efficient antimicrobial agents.

Potential aggregation hot spots in recombinant human keratinocyte growth factor; a computational study

Background: Recombinant human keratinocyte growth factor is a highly aggregation-prone therapeutic protein. The high aggregation liability of rhKGF is manifested by loss of the monomeric form of the protein and accumulation of the aggregated species even at moderate temperatures. Here, we analyzed rhKGF for its vulnerability towards aggregation by detection of aggregation-prone regions (APRs) using several sequence-based computational tools including TANGO, SolubiS, ZipperDB, AGGRESCAN, Zyggregator, Camsol, PASTA, SALSA, WALTZ, SODA, Amylpred, AMYPDB, and structure-based tools including Aggrescan3D and molecular dynamics-based spatial aggregation propensity (SAP) algorithm. Results: The sequence-based prediction of APRs in rhKGF indicated that they are mainly located at positions 10-30, 40-60, 61-66, 88-120, and 130-140 which are rich in β-branched aliphatic, hydrophobic, aromatic and Glutamine/Aspargine (Q/N) residues. Mapping on the rhKGF tertiary structure revealed that most of these residues including F16-R25, I43, E45, R47-I56, F61, Y62, N66, L88-E91, E108-F110, A112, N114, T131, and H133-T140 are surface-exposed in the natively folded protein which can promote aggregation without major unfolding event or the conformational change may occur in the oligomers composed of natively folded monomers. The other regions are buried in the native state and their contribution to non-native aggregation is mediated by a preceding unfolding event in the monomeric state of the protein. The structure-based prediction of APRs using SAP tool limited the number of identified APRs to the dynamically-exposed hydrophobic residues including V12, A50, V51, L88, I89, L90, I118, L135, and I139 mediating the native-state aggregation. Conclusion: Our analysis of APRs in rhKGF identified the regions determining the intrinsic aggregation propensity in both folded (native) and unfolded state of the protein. These regions are the candidate positions for engineering the rhKGF sequence to reduce its aggregation tendency.

Molecular Interactions between Asphaltene and Surfactants in a Hydrocarbon Solvent: Application to Asphaltene Dispersion

Heavy oil and bitumen supply the vast majority of energy resources in Canada. Different methods can be implemented to produce oil from such unconventional resources. Surfactants are employed as an additive to water/steam to improve an injected fluid’s effectiveness and enhance oil recovery. One of the main fractions in bitumen is asphaltene, which is a non-symmetrical molecule. Studies of interactions between surfactants, anionic, and non-anionic, and asphaltene have been very limited in the literature. In this paper, we employed molecular dynamics (MD) simulation to theoretically focus on the interactions between surfactant molecules and different types of asphaltene molecules observed in real oil sands. Both non-anionic and anionic surfactants showed promising results in terms of dispersant efficiency; however, their performance depends on the asphaltene architecture. Moreover, a hydrogen/carbon (H/C) ratio of asphaltenes plays an inevitable role in asphaltene aggregation behavior. A higher H/C ratio resulted in decreasing asphaltene aggregation tendency. The results of these studies will give a deep understanding of the interactions between asphaltene and surfactant molecules.