Formation Of Insoluble Aggregates Research Articles

Inhibition of the interactions of myofibrillar proteins with gallic acid by β-cyclodextrin-metal-organic frameworks improves gel quality under oxidative stress

Generally, high levels of natural phenolic antioxidants, which are needed in meat products to exert equivalent antioxidant effects as synthetic antioxidants, can interact with myofibrillar proteins and cause aggregation of the proteins, giving rise to undesirable deterioration of gel quality. In this study, β-cyclodextrin-metal-organic frameworks (K-β-CD-MOFs) were synthesized based on β-CD and K+ ions using methanol vapor diffusion method, and used to inhibit excessive covalent and non-covalent interactions between gallic acid (GA) and myofibrillar proteins under oxidative stress without degrading the phenolic antioxidant activity. Results of zeta potential, scanning electron microscope (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC) provided evidence for the formation of GA/K-β-CD-MOFs inclusion complexes. From sulfydryl content, surface hydrophobicity, tryptophan fluorescence, turbidity and particle size analyses, the presence of K-β-CD-MOFs dose-dependently prevented the sulfydryl loss, unfolding of the proteins and formation of insoluble aggregates caused by GA (180 μM/g protein). Dynamic rheology, cooking loss, gel strength, water distribution, microstructure and Raman spectrum were further tested to evaluate effects of K-β-CD-MOFs on the qualities of GA-treated myofibrillar protein gel. Results indicated that more proteins were involved in the formation of a more ordered and homogeneous gel structure with higher gel elasticity and strength, more immobilized water and decreased cooking loss. The overall gel properties of myofibrillar proteins were improved. K-β-CD-MOFs ameliorated the instability of protein structure, manifested as increase in β-sheet content and inter-chain hydrogen bonding and decrease in hydrophobic forces (p < 0.05). This paper provides a novel method to increase the loading amount of phenolic antioxidants without jeopardizing the gel quality of meat products.

Dual-environment-sensitive probe to detect protein aggregation in stressed laryngeal carcinoma cells and tissues.

The interplay between protein folding and biological activity is crucial, with the integrity of the proteome being paramount to ensuring effective biological function execution. In this study, we report a dual-environment-sensitive probe A1, capable of selectively binding to protein aggregates and dynamically monitoring their formation and degradation. Through in vitro, cellular, and tissue assays, A1 demonstrated specificity in distinguishing aggregated from folded protein states, selectively partitioning into aggregated proteins. Thermal shift assays revealed A1 could monitor the process of protein aggregation upon binding to misfolded proteins and preceding to insoluble aggregate formation. In cellular models, A1 detected stress-induced proteome aggregation in TU212 cells (laryngeal carcinoma cells), revealing a less polar microenvironment within the aggregated proteome. Similarly, tissue samples showed more severe proteome aggregation in cancerous tissues compared to paracancerous tissues. Overall, A1 represents a versatile tool for probing protein aggregation with significant implications for both fundamental research and clinical diagnostics.

Serotonin receptors - a potential target for the treatment of Alzheimer’s disease

Alzheimer’s disease (AD) is the most common cause of dementia worldwide, having an increasing impact on aging societies. It is known that the serotonin (5-HT) system of the brain, in addition to its critical role in the control of various physiological functions and behaviors, is involved in the regulation of migration, proliferation, differentiation, maturation and programmed death of neurons. At the same time, increasing evidence indicates the involvement of 5-HT neurotransmission in the mechanisms underlying the formation of insoluble aggregates of β-amyloid and tau protein, which are the main histopathological signs of AD. In this review, we focused our attention on the available data on the participation of various 5-HT receptors and the intracellular signaling cascades induced by them in pathological processes leading to the development of AD. First of all, this concerns information about the involvement of 5-HT receptors in the mechanisms of protein aggregation in AD, which indicate that specific changes in the function of certain 5-HT receptors or associated intracellular signal transduction mediators prevent the accumulation of β-amyloid plaques and neurofibrillary tau protein tangles. Based on the accumulated experimental data, it can be assumed that the use of 5-HT receptors as new drug targets may not only be useful for improving cognitive performance in AD, but will also play an important role in treating the causes of AD-related dementia.

Serotonin Receptors as a Potential Target in the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia worldwide that has an increasing impact on aging societies. Besides its critical role in the control of various physiological functions and behavior, brain serotonin (5-HT) system is involved in the regulation of migration, proliferation, differentiation, maturation, and programmed death of neurons. At the same time, a growing body of evidence indicates the involvement of 5-HT neurotransmission in the formation of insoluble aggregates of β-amyloid and tau protein, the main histopathological signs of AD. The review describes the role of various 5-HT receptors and intracellular signaling cascades induced by them in the pathological processes leading to the development of AD, first of all, in protein aggregation. Changes in the functioning of certain types of 5-HT receptors or associated intracellular signaling mediators prevent accumulation of β-amyloid plaques and tau protein neurofibrillary tangles. Based on the experimental data, it can be suggested that the use of 5-HT receptors as new drug targets will not only improve cognitive performance in AD, but will be also important in treating the causes of AD-related dementia.

Approaches to produce and characterize recombinant protein VP1-2A of HAV for serological rapid test application

Studies reporting the expression of hepatitis A virus (HAV) structural proteins, specifically recombinant VP1-2A containing an immunogenic activity, use the Escherichia coli system. Recombinant HAV proteins may represent a source of less expensive antigens for application in different diagnostic platforms. However, the formation of insoluble aggregates is an obstacle to obtaining large amounts of HAV proteins in their native form. To overcome this obstacle, some approaches were applied in this study to improve purification, solubility, and protein expression levels. Critical properties were evaluated. The introduction of another insertion codon to increase the protein concentration and vector activity was observed and verified by SDS-PAGE. The expression was established with 0.4 mM IPTG for 4 h at 37 °C. The VP1 protein was partially soluble at an isoeletric point (pI) of 6.45. The majority of HAV VP1-2A proteins measured 45.19 kDa in size and had a homogeneity of 53.58%. Multi-antigen print immunoassay (MAPIA) showed antigenicity at different HAV VP1-2A concentrations, and microsphere-based immunoassays showed a specificity of 100% and a sensitivity of 84%. HAV VP1-2A was characterized using different sensitivity methods to prove its biological activity, indicating its use as a tool for the diagnosis of Hepatitis A virus infection.

Aggregates from the concentration process for soybean protein isolate hydrolysates: Impacts, characteristics and preventive strategies

Concentrated soybean protein isolate hydrolysates (SPIH) are widely used as umami condiment. However, the formation of insoluble aggregates during the concentration affects the product quality. Thus, this study investigated the composition and interaction forces of aggregates as well as the composition and taste change of SPIH after aggregate formation. Based on the above findings, several strategies were explored to inhibit aggregate formation without compromising the taste of SPIH. Results suggested that the aggregates were primarily consisted of Tyr (72.98 g/100 g) and were formed through hydrophobic interactions. Additionally, the aggregate content was affected by concentrating conditions like temperature and pH et al. The nutritional value and flavor quality of SPIH decreased after the formation of aggregates. It was found that adding polysaccharide coagulants, especially guar gum, may inhibit aggregates formation during concentration, but also negatively impact the umami taste. Incorporating aldose could address the issue by enhancing the umami taste of SPIH. For example, the addition of a mixture of guar gum and arabinose (1:40) could effectively suppress the formation of aggregates (76.5%) and increase the umami intensity of concentrated SPIH. This paper presents a practical and efficient approach for obtaining high-quality SPIH with outstanding umami taste.

Mechanism of Insoluble Aggregate Formation in a Reconstituted Solution of Spray-Dried Protein Powder

BackgroundSpray-drying is considered a promising alternative drying method to lyophilization (freeze-drying) for therapeutic proteins. Particle counts in reconstituted solutions of dried solid dosage forms of biologic drug products are closely monitored to ensure product quality. We found that high levels of particles formed after reconstitution of protein powders that had been spray-dried under suboptimal conditions.MethodsVisible and subvisible particles were evaluated. Soluble proteins in solution before spray-drying and in the reconstituted solution of spray-dried powder were analyzed for their monomer content levels and melting temperatures. Insoluble particles were collected and analyzed by Fourier transform infrared microscopy (FTIR), and further analyzed with hydrogen-deuterium exchange (HDX).ResultsParticles observed after reconstitution were shown not to be undissolved excipients. FTIR confirmed their identity as proteinaceous in nature. These particles were therefore considered to be insoluble protein aggregates, and HDX was applied to investigate the mechanism underlying aggregate formation. Heavy-chain complementarity-determining region 1 (CDR-1) in the aggregates showed significant protection by HDX, suggesting CDR-1 was critical for aggregate formation. In contrast, various regions became more conformationally dynamic globally, suggesting the aggregates have lost protein structural integrity and partially unfolded after spray-drying.DiscussionThe spray-drying process could have disrupted the higher-order structure of proteins and exposed the hydrophobic residues in CDR-1 of the heavy chain, contributing to the formation of aggregate through hydrophobic interactions upon reconstitution of spray-dried powder. These results can contribute to efforts to design spray-dry resilient protein constructs and improve the robustness of the spray-drying process.

Maximizing cholesterol-lowering benefits of soy protein isolate by glycation with soy soluble polysaccharide

The cholesterol-lowering benefits of two by-products of soybean processing, soy protein isolate (SPI) and soy soluble polysaccharide (SSPS), have been well known in the field, but it remains to be determined whether the combined use of both ingredients can produce synergic benefiting effects. In the work, we reported that the glycation with SSPS with increasing degrees of glycation (DG) progressively improved the potential cholesterol-lowering benefits of SPI. The cholesterol-lowering benefits were evaluated with an in vitro pepsin-trypsin digestion model, by determining the cholesterol-lowering activities (including cholesterol and bile acids binding capacities, and inhibition of micellar cholesterol solubilization) of the gastrointestinal digests. As compared with the conventional heating method (at 60 °C for 7 days under controlled relative humidity conditions), the microwave treatment was much more efficient and effective to form covalent SPI-SSPS conjugates with excellent cholesterol-lowering benefits. An extensive glycation (e.g., with DG values > 40%) significantly inhibited the hydrolysis of the proteins by the proteases, and greatly facilitated the formation of insoluble aggregates during the post digestion. The remarkably improved cholesterol-lowering benefits of the extensively glycated SPIs with SSPS could be largely attributed to the enhanced tendency to form the insoluble aggregates in the digests. In contrast, the improved benefits of the moderately glycated SPIs might be mainly associated with the formation of micelle-like SPI-SSPS conjugate assemblies in the digests. This is the first report to indicate that proteins and polysaccharides may produce synergic cholesterol-lowering benefits. Considering that both SPI and SSPS are important food ingredients, the results may have great implications for the development of plant protein-based functional foods with outstanding cholesterol-lowering benefits. • The glycation with soy soluble polysaccharide greatly improved the cholesterol-lowering activities of SPI. • The microwave-assisted glycation was more effective than the conventional dry heat method. • The improved benefits of SPI were largely due to the facilitated formation of insoluble aggregates. • The synergic cholesterol-lowering effects of proteins and polysaccharides were first reported.

Refolding of Proteins Expressed as Inclusion Bodies in E. coli.

Microbial-based biotherapeutics that are produced in Escherichia coli (E. coli) can be generated intracellularly in the form of inclusion bodies (IBs) or in soluble active form in periplasmic space or extracellularly. Overexpression of these biotherapeutics in E. coli leads to formation of insoluble aggregates called inclusion bodies. These IBs contain misfolded and inactive form of proteins which need to be refolded to obtain a functionally active form of proteins. Here, we discuss refolding of E. coli-based recombinant human granulocyte colony-stimulating factor (GCSF), expressed as IBs, and highlight some of the key features associated with the refolding kinetic reaction.

Reduction of kinesin I heavy chain decreases tau hyperphosphorylation, aggregation, and memory impairment in Alzheimer's disease and tauopathy models.

Many neurodegenerative diseases, such as Alzheimer's disease (AD) and frontotemporal dementia with Parkinsonism linked to chromosome 17, are characterized by tau pathology. Numerous motor proteins, many of which are involved in synaptic transmission, mediate transport in neurons. Dysfunction in motor protein-mediated neuronal transport mechanisms occurs in several neurodegenerative disorders but remains understudied in AD. Kinesins are the most important molecular motor proteins required for microtubule-dependent transport in neurons, and kinesin-1 is crucial for neuronal transport among all kinesins. Although kinesin-1 is required for normal neuronal functions, the dysfunction of these motor domains leading to neurodegenerative diseases is not fully understood. Here, we reported that the kinesin-I heavy chain (KIF5B), a key molecular motor protein, is involved in tau homeostasis in AD cells and animal models. We found that the levels of KIF5B in P301S tau mice are high. We also found that the knockdown and knockout (KO) of KIFf5B significantly decreased the tau stability, and overexpression of KIF5B in KIF5B-KO cells significantly increased the expression of phosphorylated and total tau levels. This suggested that KIF5B might prevent tau accumulation. By conducting experiments on P301S tau mice, we showed that partially reducing KIF5B levels can reduce hyperphosphorylation of the human tau protein, formation of insoluble aggregates, and memory impairment. Collectively, our results suggested that decreasing KIF5B levels is sufficient to prevent and/or slow down abnormal tau behavior of AD and other tauopathies.

Atypical Ubiquitination and Parkinson's Disease.

Ubiquitination (the covalent attachment of ubiquitin molecules to target proteins) is one of the main post-translational modifications of proteins. Historically, the type of polyubiquitination, which involves K48 lysine residues of the monomeric ubiquitin, was the first studied type of ubiquitination. It usually targets proteins for their subsequent proteasomal degradation. All the other types of ubiquitination, including monoubiquitination; multi-monoubiquitination; and polyubiquitination involving lysine residues K6, K11, K27, K29, K33, and K63 and N-terminal methionine, were defined as atypical ubiquitination (AU). Good evidence now exists that AUs, participating in the regulation of various cellular processes, are crucial for the development of Parkinson’s disease (PD). These AUs target various proteins involved in PD pathogenesis. The K6-, K27-, K29-, and K33-linked polyubiquitination of alpha-synuclein, the main component of Lewy bodies, and DJ-1 (another PD-associated protein) is involved in the formation of insoluble aggregates. Multifunctional protein kinase LRRK2 essential for PD is subjected to K63- and K27-linked ubiquitination. Mitophagy mediated by the ubiquitin ligase parkin is accompanied by K63-linked autoubiquitination of parkin itself and monoubiquitination and polyubiquitination of mitochondrial proteins with the formation of both classical K48-linked ubiquitin chains and atypical K6-, K11-, K27-, and K63-linked polyubiquitin chains. The ubiquitin-specific proteases USP30, USP33, USP8, and USP15, removing predominantly K6-, K11-, and K63-linked ubiquitin conjugates, antagonize parkin-mediated mitophagy.

Sirtuin-1 sensitive lysine-136 acetylation drives phase separation and pathological aggregation of TDP-43

Trans-activation response DNA-binding protein of 43 kDa (TDP-43) regulates RNA processing and forms neuropathological aggregates in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Investigating TDP-43 post-translational modifications, we discovered that K84 acetylation reduced nuclear import whereas K136 acetylation impaired RNA binding and splicing capabilities of TDP-43. Such failure of RNA interaction triggered TDP-43 phase separation mediated by the C-terminal low complexity domain, leading to the formation of insoluble aggregates with pathologically phosphorylated and ubiquitinated TDP-43. Introduction of acetyl-lysine at the identified sites via amber suppression confirmed the results from site-directed mutagenesis. K84-acetylated TDP-43 showed cytoplasmic mislocalization, and the aggregation propensity of K136-acetylated TDP-43 was confirmed. We generated antibodies selective for TDP-43 acetylated at these lysines, and found that sirtuin-1 can potently deacetylate K136-acetylated TDP-43 and reduce its aggregation propensity. Thus, distinct lysine acetylations modulate nuclear import, RNA binding and phase separation of TDP-43, suggesting regulatory mechanisms for TDP-43 pathogenesis.

Emerging Roles for Phase Separation of RNA-Binding Proteins in Cellular Pathology of ALS.

Liquid-liquid phase separation (LLPS) is emerging as a major principle for the mesoscale organization of proteins, RNAs, and membrane-bound organelles into biomolecular condensates. These condensates allow for rapid cellular responses to changes in metabolic activities and signaling. Nowhere is this regulation more important than in neurons and glia, where cellular physiology occurs simultaneously on a range of time- and length-scales. In a number of neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS), misregulation of biomolecular condensates leads to the formation of insoluble aggregates—a pathological hallmark of both sporadic and familial ALS. Here, we summarize how the emerging knowledge about the LLPS of ALS-related proteins corroborates with their aggregation. Understanding the mechanisms that lead to protein aggregation in ALS and how cells respond to these aggregates promises to open new directions for drug development.

Evaluation of different expression systems for the heterologous production of sprivivirus glycoprotein

Glycoprotein mediated the entry of rhabdoviruses into the host cell and was the target of neutralizing antibodies. It was a prime tool for the study of the evolution and pathogenesis of viruses. Here, four different expression systems including cell-free synthesis system, Escherichia coli, Pichia pastoris and the baculovirus infected Spodoptera frugiperda were used to explore the recombinant expression of spring viraemia of carp virus glycoprotein. The results indicated that the recombinant glycoprotein could be detected in E. coli cell-free system after in vitro incubation for 16 h at 25 °C. E. coli was found to be the optimal expression system for the production of the largest amount of recombinant glycoprotein. Although robust yields were considerable in E. coli, the formation of insoluble aggregates and the removal of solubilizing labels were challenging for the expression and purification of glycoprotein. For this reason, the higher eukaryotic expression systems, yeast GS115 and insect Sf9 cells were used for the production of glycoprotein. The purity of the recombinant protein obtained from yeast culture was over 90% after purification by Ni-NTA. In insect cells, the target protein was expressed mainly in the intracellular environment, but secretory glycoprotein was also detected by western blotting. These findings provided a basis for further investigations into the structure and function of SVCV glycoprotein.

Correlation between 18F-THK5351 tau PET imaging and the progression of neuropathology and clinical signs in a mouse model.

Several common neurodegenerative disorders are characterized by the intracellular accumulation of neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated tau protein (ptau). In Alzheimer's disease (AD), NFTs are accompanied by extracellular amyloid-beta (Aβ), but primary tauopathy disorders are marked by the accumulation of tau protein alone. Abnormal ptau leads to the formation of insoluble aggregates in tauopathies such as AD. Positron emission tomography (PET) imaging is a promising avenue that may identify tau aggregates in vivo cross-sectionally and longitudinally in various dementia conditions. In this study, tau transgenic P301S mice, from 6 to 12 months of age, were analyzed by longitudinal in vivo PET brain imaging utilizing the tau ligand [18 F]THK5351, as well as histological, biochemical, and behavioral characterization over time. Our results demonstrate elevated in vivo [18 F]-THK5351 PET signaling over time in transgenic P301S tau mice from 8 months that had a positive correlation with histological and biochemical tau changes, and motor, memory and learning impairment. This study indicates that [18 F]-THK5351 may facilitate detection of aberrant tau aggregation and deposition in order to differentiate between tauopathies, gain insights into their underlying pathophysiologies, and to have a reliable biomarker to follow during treatment trials. This work was partially funded by Department of Defense Peer Reviewed Alzheimer's Research Program Convergence Science. Research Award grant AZ160106 and Alzheimer's Association New Investigator Research Grant NIRG-394284 to IMG.

Effect of high pressure homogenization on aggregation, conformation, and interfacial properties of bighead carp myofibrillar protein.

Fish myofibrillar protein is underutilized due to the formation of insoluble aggregates in low salt media. High pressure homogenization (HPH) at 20, 40, and 60MPa for four passes was applied on bighead carp myofibrillar protein in order to modify its structure and interfacial properties. Changes in aggregation, conformation, solubility, emulsifying and foaming properties of myofibrillar protein were investigated. The aggregates of myofibrillar protein were obviously disrupted by HPH treatment. The size of myofibrillar protein aggregates became smaller and more uniform as the treating pressure increased, accompanied by notable decreases of cross-sectional height and Rq value in AFM image. Furthermore, the conformation of HPH-treated myofibrillar protein was unfolded into a flexible and open structure. α-helix and β-sheet were converted into β-turn and random coil. Surface hydrophobicity and zeta potential were strengthened, along with the exposure of sulfhydryl groups onto molecule surface. On the other hand, solubility, emulsifying activity index (EAI) and foaming capacity (FC) of HPH-treated myofibrillar protein were markedly enhanced with the increasing pressure. Especially after HPH treatment at 60MPa, myofibrillar protein was almost dissolved in low salt media (solubility 91.86%) with 4.92 fold for EAI and 3.52 fold for FC. But there was little variation in emulsifying and foaming stabilities. These results suggested that HPH treatment has interesting potential to induce the dissociation and unfolding of myofibrillar protein in low salt media, therefore improving its interfacial properties. PRACTICAL APPLICATION: Carp myofibrillar protein was treated by high pressure homogenization (HPH). Aggregates of myofibrillar protein were disrupted into smaller size form. Conformation of myofibrillar protein was unfolded into open and loose structure. Emulsifying and foaming capacities of myofibrillar protein were improved. HPH treatment modified the structure and interfacial properties of myofibrillar protein.

A Solvatochromic Fluorescent Probe Reveals Polarity Heterogeneity upon Protein Aggregation in Cells

AbstractWe report a crystallization‐induced emission fluorophore to quantitatively interrogate the polarity of aggregated proteins. This solvatochromic probe, namely “AggRetina” probe, inherently binds to aggregated proteins and exhibits both a polarity‐dependent fluorescence emission wavelength shift and a viscosity‐dependent fluorescence intensity increase. Regulation of its polarity sensitivity was achieved by extending the conjugation length. Different proteins bear diverse polarity upon aggregation, leading to different resistance to proteolysis. Polarity primarily decreases during protein misfolding but viscosity mainly increases upon the formation of insoluble aggregates. We quantified the polarity of aggregated protein‐of‐interest in live cells via HaloTag bioorthogonal labeling, revealing polarity heterogeneity within cellular aggregates. The enriched micro‐environment details inside misfolded and aggregated proteins may correlate to their bio‐chemical properties and pathogenicity.

A Solvatochromic Fluorescent Probe Reveals Polarity Heterogeneity upon Protein Aggregation in Cells.

We report a crystallization-induced emission fluorophore to quantitatively interrogate the polarity of aggregated proteins. This solvatochromic probe, namely "AggRetina" probe, inherently binds to aggregated proteins and exhibits both a polarity-dependent fluorescence emission wavelength shift and a viscosity-dependent fluorescence intensity increase. Regulation of its polarity sensitivity was achieved by extending the conjugation length. Different proteins bear diverse polarity upon aggregation, leading to different resistance to proteolysis. Polarity primarily decreases during protein misfolding but viscosity mainly increases upon the formation of insoluble aggregates. We quantified the polarity of aggregated protein-of-interest in live cells via HaloTag bioorthogonal labeling, revealing polarity heterogeneity within cellular aggregates. The enriched micro-environment details inside misfolded and aggregated proteins may correlate to their bio-chemical properties and pathogenicity.

Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens

Bacterial expression systems remain a widely used host for recombinant protein production. However, overexpression of recombinant target proteins in bacterial systems such as Escherichia coli can result in poor solubility and the formation of insoluble aggregates. As a consequence, numerous strategies or alternative engineering approaches have been employed to increase recombinant protein production. In this case study, we present the strategies used to increase the recombinant production and solubility of ‘difficult-to-express’ bacterial antigens, termed Ant2 and Ant3, from Absynth Biologics Ltd.’s Clostridium difficile vaccine programme. Single recombinant antigens (Ant2 and Ant3) and fusion proteins (Ant2-3 and Ant3-2) formed insoluble aggregates (inclusion bodies) when overexpressed in bacterial cells. Further, proteolytic cleavage of Ant2-3 was observed. Optimisation of culture conditions and changes to the construct design to include N-terminal solubility tags did not improve antigen solubility. However, screening of different buffer/additives showed that the addition of 1–15 mM dithiothreitol alone decreased the formation of insoluble aggregates and improved the stability of both Ant2 and Ant3. Structural models were generated for Ant2 and Ant3, and solubility-based prediction tools were employed to determine the role of hydrophobicity and charge on protein production. The results showed that a large non-polar region (containing hydrophobic amino acids) was detected on the surface of Ant2 structures, whereas positively charged regions (containing lysine and arginine amino acids) were observed for Ant3, both of which were associated with poor protein solubility. We present a guide of strategies and predictive approaches that aim to guide the construct design, prior to expression studies, to define and engineer sequences/structures that could lead to increased expression and stability of single and potentially multi-domain (or fusion) antigens in bacterial expression systems.

RNase P Inhibitors Identified as Aggregators.

RNase P is an essential enzyme responsible for tRNA 5'-end maturation. In most bacteria, the enzyme is a ribonucleoprotein consisting of a catalytic RNA subunit and a small protein cofactor termed RnpA. Several studies have reported small-molecule inhibitors directed against bacterial RNase P that were identified by high-throughput screenings. Using the bacterial RNase P enzymes from Thermotoga maritima, Bacillus subtilis, and Staphylococcus aureus as model systems, we found that such compounds, including RNPA2000 (and its derivatives), iriginol hexaacetate, and purpurin, induce the formation of insoluble aggregates of RnpA rather than acting as specific inhibitors. In the case of RNPA2000, aggregation was induced by Mg2+ ions. These findings were deduced from solubility analyses by microscopy and high-performance liquid chromatography (HPLC), RnpA-inhibitor co-pulldown experiments, detergent addition, and RnpA titrations in enzyme activity assays. Finally, we used a B. subtilis RNase P depletion strain, whose lethal phenotype could be rescued by a protein-only RNase P of plant origin, for inhibition zone analyses on agar plates. These cell-based experiments argued against RNase P-specific inhibition of bacterial growth by RNPA2000. We were also unable to confirm the previously reported nonspecific RNase activity of S. aureus RnpA itself. Our results indicate that high-throughput screenings searching for bacterial RNase P inhibitors are prone to the identification of "false positives" that are also termed pan-assay interference compounds (PAINS).