Single-step purification and characterization of Pseudomonas aeruginosa azurin.

Azurin is a small periplasmic blue copper protein found in bacterial strains such as Pseudomonas and Alcaligenes where it facilitates denitrification. Azurin is extensively studied for its ability to mediate electron-transfer processes, but it has also sparked interest of the pharmaceutical community as a potential antimicrobial or anticancer agent. Here we offer a novel approach for expression and single-step purification of azurin in Escherichia coli with high yields and optimal metalation. A fusion tag strategy using an N-terminal GST tag was employed to obtain pure protein without requiring any additional purification steps. After the on-column cleavage by HRV 3C Protease, azurin is collected and additionally incubated with copper sulphate to ensure sufficient metalation. UV-VIS absorption, mass spectroscopy, and circular dichroism analysis all validated the effective production of azurin, appropriate protein folding and the development of an active site with an associated cofactor. MD simulations verified that incorporation of the N-terminal GPLGS segment does not affect azurin structure. In addition, the biological activity of azurin was tested in HeLa cells.

Removal of tritiated water molecules by isotope exchange reaction between H2O vapor and tritium water.

Developing a cost-effective method for separating and concentrating tritium water (HTO) from light water (H2O) without consuming additional energy is crucial for achieving reliable and safe nuclear fission and fusion energy technologies. However, this presents a significant challenge because of the difficulties in obtaining basic information, such as the chemical and physical properties of HTO molecules. Here, we investigate the isotope exchange reaction (IER) between HTO molecules in H2O solution and H2O vapor in the atmosphere. The reduction and purification rates of HTO-containing water were measured by varying the system conditions, such as temperature (20-50 °C) and humidity (50 %-90 %), under an equilibrium state between the liquid phase (water) and vapor phase (air). Our findings indicate that the concentration of HTO in the solution can be significantly reduced by increasing H2O vapor in the atmosphere. This result can be quantitatively explained by considering the entropy of mixing between the solution and vapor phases. The results obtained here provide both basic understanding on the exchange process between liquid- and vapor-water molecules and a passive technology for treating HTO-containing water.

Enhancing monoclonal antibody stability during protein a chromatography using 2-methyl imidazolium dihydrogen phosphate.

This study investigates the impact of 2-methyl imidazolium dihydrogen phosphate (2-MIDHP) on monoclonal antibody (mAb) aggregation during the Protein A purification stage, at a low pH (pH 3.0), and the viral inactivation phase. Size-exclusion high-performance liquid chromatography (SE-HPLC) and dynamic light scattering (DLS) were used to assess the mAb aggregation. Additionally, the influence of 2-MIDHP on mAb recovery, host cell protein (HCP) clearance, and Protein A leaching was investigated. Thermal stability of mAb, eluted in buffers containing 5 % to 25 % 2-MIDHP was analysed, using differential scanning calorimetry (DSC). Structural insights were obtained via circular dichroism (CD) and fluorescence spectroscopy. Our findings indicated that 2-MIDHP exerted a concentration-dependent protective effect against mAb aggregation, at the pH of 3.0. As the concentration of 2-MIDHP was increased from 0 % to 25 %, the aggregation was significantly reduced from 3.8 ± 0.01 % to 0.56 ± 0.002 %, as analysed by SE-HPLC. Addition of 2-MIDHP did not significantly impact the mAb recovery, HCP clearance, or Protein A leaching. DSC data supported these results, with higher 2-MIDHP concentrations leading to increased melting temperatures of mAb. CD and fluorescence spectroscopy revealed no significant changes in the secondary structure or aromatic residue environment in 2-MIDHP-treated samples, despite the observed reduction in aggregation. The results suggested that 2-MIDHP mitigated mAb aggregation during Protein A purification, possibly by stabilizing the protein structure under acidic stress conditions. These findings offer valuable insights for improving the robustness of mAb purification processes, enhancing product quality and yield.

Heterologous expression and purification of the phage lysin-like bacteriocin LysL from lactococcus lactis LAC460.

The wild-type Lactococcus lactis strain LAC460 produces two bacteriocin-like phage lysins, LysL and LysP. This study aimed to produce and secrete LysL in various heterologous hosts and an In vitro cell-free expression system for further functional studies. Initially, the lysL gene from L. lactis LAC460 was cloned into Lactococcus cremoris NZ9000 and L. lactis N8 strains, with and without the usp45 signal sequence (SSusp45), under a nisin-inducible promoter. Active LysL was primarily produced intracellularly in recombinant L. lactis N8, with some secretion into the supernatant. Recombinant L. cremoris NZ9000 lysed upon nisin induction, indicating successful lysL expression. However, fusion with Usp45 signal peptide (SPUsp45-LysL) weakened LysL activity, likely due to incomplete signal peptide cleavage during secretion. Active LysL was also produced In vitro, and analysed in SDS-PAGE, giving a 42 kDa band. However, the yield of LysL protein was still low when produced from recombinant lactococci or by In vitro expression system. Therefore, His-tagged LysL was produced in Escherichia coli BL21(DE3). Western blot confirmed the intracellular production of about 44-kDa His-tagged LysL in E. coli. His-tagged active LysL was then purified by Ni-NTA affinity chromatography yielding sufficient 4.34 mg of protein to be used in future functional studies.

Heterologous expression, characterization, and application of recombinant thermostable α-amylase from Geobacillus sp. DS3 for porous starch production.

Novel Geobacillus sp. DS3, isolated from the Sikidang Crater in Dieng, exhibits promising characteristics for industrial applications, particularly in thermostable α-amylase production. Recombinant technology was used to express thermostable α-amylase in E. coli BL21(DE3) to overcome high-temperature production challenges. The study aimed to express, purify, characterize, and explore potential applications of this novel enzyme. The enzyme was successfully expressed in E. coli BL21(DE3) at 18 °C for 20 h with 0.5 mM IPTG induction. Purification with Ni-NTA column yielded 69.23 % from the initial crude enzyme, with a 3.6-fold increase in specific activity. The enzyme has a molecular weight of ±70 kDa (±58 kDa enzyme+11 kDa SUMO protein). It exhibited activity over a wide temperature range (30-90 °C) and pH range (6-8), with optimal activity at 70 °C and pH 6 with great stability at 60 °C. Kinetic analysis revealed Km and Vmax values of 324.03 mg/ml and 36.5 U/mg, respectively, with dextrin as the preferred substrate without cofactor addition. As a metalloenzyme, it showed the best activity in the presence of Ca2+. The enzyme was used for porous starch production and successfully immobilized with chitosan, exhibiting improved thermal stability. After the fourth reuse, the immobilized enzyme maintained 62 % activity compared to the initial immobilization.

Protocol for the production and reconstitution of VDAC1 for functional assays.

The voltage-dependent anion channel (VDAC) is an abundant and multifunctional outer mitochondrial membrane protein, playing key roles in neurodegeneration, apoptosis, and mitochondrial membrane biogenesis. Here, we present a protocol to produce and reconstitute high yields of detergent-solubilized VDAC, expressed as inclusion bodies in E. coli. We describe steps for purification by affinity chromatography and refolding in lauryldimethylamine-N-oxide (LDAO). We then detail procedures for reconstituting VDAC into membrane vesicles to assay its channel and phospholipid scramblase activity via fluorescence-based assays. For complete details on the use and execution of this protocol, please refer to Bergdoll et al.,1 Queralt-Martín et al., 2 and Jahn et al.3.

A validated UPLC-MS/MS method for the quantification of immunosuppressive drugs in peripheral blood mononuclear cells using liquid-liquid extraction with low temperature purification without complex pretreatment steps.

Immunosuppressive drugs (ISDs) are given to avoid the allograft rejection after transplantation. The concentrations of ISDs should be closely monitored owing to their wide inter-individual variability in its pharmacokinetics and narrow therapeutic window. Currently, the whole blood concentration measurement is the major approach of therapeutic drug monitoring of clinical ISDs in organ transplantation. Its correlation with the efficacy of ISDs remains elusive. While the acute rejection after transplantation may occur even when whole-blood ISDs concentrations are within the target range. Since the site of action of ISDs are within the lymphocyte, direct measurement of drug exposure in target cells may more accurately reflect the clinical efficacy of ISDs. Although several methods have been developed for the peripheral blood mononuclear cells (PBMCs) extraction and drug concentration measurement, the complex pre-processing has limited the study of the relationship between intracellular ISDs concentrations and the occurrence of rejection. In this study, the extraction of ISDs in PBMCs was carried out by the liquid-liquid extraction with low temperature purification, without centrifugation. The lower limit of quantitation were 0.2 ng/mL for cyclosporine A, tacrolimus and sirolimus, 1.0 ng/mL for mycophenolic acid, and the within-run and between-run coefficient of variations were both less than 12.4 %. The calibration curves of mycophenolic acid had a linear range (ng/mL): 1.0-128.0 (r2 = 0.9992). The calibration curves of other three ISDs had a linear range (ng/mL): 0.2-20.48 (r2 > 0.9956). A total of 157 clinical samples were analyzed by the UPLC-MS/MS for ISDs concentration in blood or plasma ([ISD]blood or plasma) and the concentration within PBMCs ([ISD]PBMC). Although there was strong association between [ISD]PBMC and [ISD]blood or plasma, the large discrepancies between concentration within [ISD]blood or plasma and [ISD]PBMC were observed in a small proportion of clinical samples. The developed method with short analysis time and little amounts of blood sample can be successfully applied to therapeutic drug monitoring of ISDs in PBMCs for analysis of large numbers of clinical samples and is helpful to explore the clinical value of ISDs concentration in PBMCs.

The [(bathophenanthroline)3:Fe2+] complex as an aromatic non-polymeric medium for purification of human lactoferrin.

We describe a non-chromatographic, ligand-free platform for the efficient purification of recombinant human lactoferrin (LF). The platform consists of a [metal:chelator] complex precipitate in the presence of osmotically active polyethylene glycol 6000 (PEG-6000). Purification is achieved in three stages. Following formation of the complex, LF is captured under neutral conditions by the aggregated complexes (Step I), a washing step follows (Step II) and then, (Step III) LF is extracted in pure form with 100 mM tribasic Na citrate buffer (pH 7). Of the four complexes investigated, [bathophenanthroline (batho)3:Fe2+] was determined to be the most efficient. LF is recovered with high yield (∼90%, by densitometry) and purity (≥97%, by SDS polyacrylamide gel electrophoresis (SDS-PAGE)) from an artificial contamination background comprising E. coli lysate proteins. Purified LF is demonstrated to be monomeric by dynamic light scattering (DLS); to preserve its native secondary structure by circular dichroism (CD) spectroscopy; and, as apo-LF, to efficiently inhibit bacterial growth. Process yield is not affected by a 45-fold increase in LF concentration from 0.2 to 9 mg/mL. We provide evidence that protein capture relies on [cation:π] interactions between the lysine and arginine residues of LF with the fully aromatic [(batho)3:Fe2+] complexes. The use of [metal:chelator] complex aggregates is demonstrated to provide an economical and efficient avenue for LF purification.

A review of Lycium barbarum polysaccharides: Extraction, purification, structural-property relationships, and bioactive molecular mechanisms.

Lycium barbarum L. is of great significance medicinal and edible plant, which is native to N. & Central China. The extensive health benefits of L. barbarum have earned it great respect in traditional medicine for centuries. Lycium barbarum polysaccharides (LBPs) being recognized as one of the most crucial bioactive compounds found within this plant, with it exhibit a diverse range of pharmacological activities and nutritional functions, thereby generating substantial market demand and broad application prospects. To gain a more comprehensive understanding of LBPs, the review discussed the extraction, purification and structural-property relationships of these compounds. In addition, this review provides a comprehensive summary of the potential mechanisms underlying various biological activities attributed to LBPs, including immune modulation, antioxidant effects, neuroprotection, hepatoprotection, and antitumor properties. The application status and the future research directions of LBPs were subsequently presented. This review will establish a robust foundation and serve as an invaluable resource for future research and advancements in the field of LBPs.

Macroporous polymeric resin for the purification of flavonoids from medicinal plants: A review.

The purification of flavonoids using the macroporous polymer resin method has gained attention in recent years due to its simplicity, precision, cost-effectiveness, and the ability to separate flavonoids from other constituents. Several studies have been conducted to investigate the efficiency and effectiveness of macroporous polymer resin in purifying flavonoids from various plant sources. This review aims to evaluate the existing literature on macroporous polymer resin purification of flavonoids and provide a comprehensive analysis of the current research trends and advancements in this field. It also highlights the importance of optimizing the adsorption parameters and conditions such as resin type, resin concentration, pH, and temperature for efficient purification of flavonoids using macroporous polymer resin. The key findings of this review reveal that macroporous resins with weak polarity, large surface areas, and pore diameters have a stronger adsorption capacity for flavonoids compared to polar resins. Furthermore, ultrasonic-solvent assisted extraction often combines with macroporous resin for effective the extraction and purification of flavonoids.

Expression of F1L, a vaccinia virus H3L transmembrane protein analogue of orf virus, and its successful purification as a diagnostic antigen.

Orf or contagious ecthyma is a highly contagious, zoonotic, and economically important global viral disease of small ruminants and is endemic in India. Vaccination of susceptible goats/sheep along with suitable recombinant protein-based serological assay will be useful in the control of the infection. In this study, the full-length and truncated versions of F1L encoding gene (ORF 059) of orf virus were cloned into pFasBac HT A vector, transformed in DH10Bac cells, and expressed in insect cells. The full-length and truncated recombinant F1L proteins were expressed as a 6 × histidine-tagged fusion protein for ease of purification by Ni-NTA affinity chromatography under denaturing conditions. A protein with ~ 40 kDa and ~ 35 kDa for full-length and truncated F1L protein, respectively, were expressed and confirmed by SDS-PAGE and western blot. The protein reactivity evaluated by western blot analysis and indirect ELISA using ORFV hyperimmune serum was also found to be reactive. The results of the present study showed that the purified recombinant F1L protein can be used as a diagnostic antigen in sero-surveillance of ORFV infection in small ruminants. To the best of authors' knowledge, this is the first report on the expression of ORFV F1L in insect cells using a baculovirus vector and its successful purification to use as the potential diagnostic antigen in ELISA.

Human Circulatory/Respiratory-Inspired Comprehensive Air Purification System.

The circulatory and respiratory systems in humans are marvels of biological engineering that exhibit competence in maintaining homeostasis. These systems not only shield the organism from external contaminants but also orchestrate the vital gases via the bloodstream to sustain cellular respiration and metabolic processes across diverse tissues. It is noticed that spaces inhabited encounter challenges akin to those of the human body: protecting the indoor air from external pollutants while removing anthropogenic byproducts like carbon dioxide (CO2), particulate matters (PM), and volatile organic compounds (VOCs) tooutside. A biomimetic approach, composed of a microbubble-based gas exchanger and circulating liquid inspired by alveoli, capillary beds, and bloodstream of the human circulatory/respiratory system, offer an innovative solution for comprehensive air purification of hermetic spaces. Circulatory/respiratory-inspired air purification system (CAPS) ensure both continuous removal of PM and exchange of gas species between indoor and outdoor environments to maintain homeostasis. The effectiveness of this system is also supported by animal behavior experiments with and without CAPS, showing an effect of reducing CO2 concentration by 30% and increasing mice locomotor activity by 53%. CAPS is expected to evolve into robust and comprehensive air purification schemes through the networked integration of plural internal and external environments.

Comparative Analysis of Laboratory-Scale Immunoglobulin G Purification Methods from Human Serum.

Immunoglobulin G (IgG) purification is a critical process for evaluating its role in autoimmune diseases, which are defined by the occurrence of autoantibodies. Affinity chromatography with protein G is widely considered to be the optimal technique for laboratory-scale purification. However, this technique has some limitations, including the exposure of IgG to low pH, which can compromise the quality of the purified IgG. Here, we show that alternative methods for IgG purification are possible while maintaining the quality of IgG. Different techniques for IgG purification from serum were evaluated and compared with protein G-based approaches: Melon Gel, caprylic acid-ammonium sulfate (CAAS) precipitation, anion-exchange chromatography with diethylamino ethyl (DEAE) following ammonium sulfate (AS) precipitation, and AS precipitation alone. The results demonstrated that the purification yield of these techniques surpassed that of protein G. However, differences in the purity of IgG were observed using GeLC-MS/MS. The avidity of purified IgG against selected targets (SARS-CoV-2 and topoisomerase-I) was similar between purified IgG obtained using all techniques and unpurified sera. Our work provides valuable insights for future studies of IgG function by recommending alternative purification methods that offer advantages in terms of yield, time efficiency, cost-effectiveness, and milder pH conditions than protein G.

Methods for Bioinformatic Prediction of Genuine sRNAs from Outer Membrane Vesicles.

The molecular pathogenesis of Gram-negative bacteria remains a complex and incompletely understood phenomenon. Various factors are believed to contribute to the pathogenicity of these bacteria. One key mechanism utilized by Gram-negative bacteria is the production of outer membrane vesicles (OMVs), which are small spherical particles derived from the bacterial outer membrane. These OMVs are crucial in delivering virulence factors to the host, facilitating host-pathogen interactions. Within these OMVs, small regulatory RNAs (sRNAs) have been identified as important players in modulating the host immune response. One of the main challenges in studying OMVs and their cargo of sRNAs is the difficulty in isolating and purifying sufficient quantities of OMVs, as well as accurately predicting genuine sRNAs computationally. In this chapter, we present protocols aimed at overcoming these obstacles.

Separation of Bacterial Extracellular Vesicles via High-Performance Anion Exchange Chromatography.

Bacterial extracellular vesicles (BEVs) have extraordinary biotechnological potential, but traditional purification methods lack desirable scalability and commonly co-isolate protein impurities, limiting clinical translation. Anion exchange chromatography (AEC) separates molecules based on differences in net charge and is widely used for industrial biomanufacturing of protein therapeutics. Recently, AEC has recently been applied for purification of EVs from both mammalian and bacterial sources. Since most bacteria produce BEVs with a negative surface membrane change, AEC can potentially be widely used for BEV purification. Here, we describe a method utilizing high-performance AEC (HPAEC) in tandem with size-based tangential flow filtration for improved BEV purification. We have previously found this method can reduce co-isolated protein impurities and potentiate anti-inflammatory bioactivity of probiotic BEVs. Thus, this method holds promise as a scalable alternative for improved BEV purification.

Engineering Outer Membrane Vesicles to Carry Enzymes: Encapsulation, Isolation, Characterization, and Modification.

Outer membrane vesicles (OMVs) are small, spherical, nanoscale proteoliposomes released from Gram-negative bacteria that play an important role in cellular defense, pathogenesis, and signaling, among other functions. The functionality of OMVs can be enhanced by engineering developed for biomedical and biochemical applications. Here, we describe methods for directed packaging of enzymes into bacterial OMVs of E. coli using engineered molecular systems, such as localizing proteins to the inner or outer surface of the vesicle. Additionally, we detail some modification strategies for OMVs such as lyophilization and surfactant conjugation that enable the protection of activity of the packaged enzyme when exposed to non-physiological conditions such as elevated temperature, organic solvents, and repeated freeze/thaw that otherwise lead to a substantial loss in the activity of the free enzyme.

Purification and Cryo-Electron Microscopy Analysis of Bacterial Appendages.

A number of extracellular helical protein polymers are crucial for supporting bacterial motility. The bacterial flagellum is a polymeric appendage used to support cellular motility. Historically, structural studies of flagellar and other filaments were limited to those present as or locked into straightened states. Here, we present a robust workflow that produces biologically relevant high-resolution cryo-electron microscopy (cryo-EM) structures of bacterial flagellar filaments. We highlight how a simple purification method, centered around several centrifugation steps, exploits the process of filament ejection in Caulobacter crescentus and results in isolated filaments amenable to transmission electron microscopy (TEM) studies. The quality of the sample is validated by SDS-PAGE and negative stain TEM analysis before a sample is vitrified for cryogenic electron microscopy (cryo-EM) data collection. We provide a detailed protocol for reconstructing either straight or curved flagellar filaments by cryo-EM helical reconstruction methods, followed by an overview of model building and validation. In our hands, this workflow resulted in several flagellar structures below 3 Å resolution, with one data set reaching a global resolution of 2.1 Å. The application of this workflow supports structure-function studies to better understand the molecular interactions that regulate filament architecture in biologically relevant states. Future work will not only examine interactions that regulate bacterial flagellar and other filament organization but also provide a foundation for developing new helical biopolymers for biotech applications. Key features • Rapid high-quality purification of bacterial flagella via simple bacterial culturing, centrifugation, and resuspension methods. • High-throughput cryo-EM data collection of filamentous objects. • Use of cryoSPARC implementations of helical reconstruction algorithms to generate high-resolution 3D structures of bacterial flagella or other helical polymers.

Synergistic C2H2 Binding Sites in Hydrogen-Bonded Supramolecular Framework for One-step C2H4 Purification from Ternary C2 Mixture.

Purification of C2H4 from the ternary C2 hydrocarbon mixture in one step is of critical significance but still extremely challenging according to its intermediate physical properties between C2H6 and C2H2. Hydrogen-bonded organic frameworks (HOFs) stabilized by supramolecular interactions are emerging as a new kind of adsorbents that facilitate green separation. However, it remains a problem to efficiently realize the one-step C2H4 purification from C2H6/C2H4/C2H2 mixture because of the low C2H2/C2H4 selectivity. We herein report a robust microporous HOF (termed as HOF-TDCPB) with dense O atoms and aromatic rings distributed on the pore surface which provide C2H6 and C2H2 preferred environment simultaneously. Dynamic breakthrough experiments indicate that HOF-TDCPB can not only obtain high-purity C2H4 from binary C2 mixture, but also firstly realize one-step C2H4 purification from ternary C2H6/C2H4/C2H2 mixture, with the C2H4 productivity of 3.2 L/kg (>99.999%) for one breakthrough cycle. Furthermore, HOF-TDCPB displays outstanding stability in air, organic solvents and water, which endow it excellent cycle performance even under high-humidity conditions. Theoretical calculations indicate that multiple O sites on pore channels can create synergistic binding sites for C2H2, thus affording overall stronger multipoint interactions.

A comprehensive review on fructosyl peptide oxidase as an important enzyme for present hemoglobin A1c assays.

Glycated proteins are generated by binding of glucose to the proteins in blood stream through a nonenzymatic reaction. Hemoglobin A1c (HbA1c) is a glycated protein with glucose at the N-terminal of ß-chain. HbA1c is extensively used as an indicator for assessing the blood glucose concentration in diabetes patients. There are different conventional clinical methods for the detection of HbA1c. However, enzymatic detection method has newly obtained great attention for its high precision and cost-effectiveness. Today, fructosyl peptide oxidase (FPOX) plays a key role in the enzymatic measurement of HbA1c, and different companies have marketed HbA1c assay systems based on FPOX. Recent investigations show that FPOX could be used in assaying HbA1 without requiring HbA1c primary digestion. It could also be applied as a biosensor for HbA1c detection. In this review, we have discussed the recent improvements of FPOX properties, different methods of FPOX purification, solubility, and immobilization, and also the use of FPOX in HbA1c biosensors.

Interfacial modification of recombinant protein for immunoglobulin G adsorption with spindle-shaped MOF as nano molecular containers.

Development of fresh solid phase extractant is critical for selective separation and purification of special proteins. Herein, we demonstrated a recombinant Staphylococcal Protein G (rSPG) with a His-tag modified the novel single-metal organic framework (rSPG@Ni-MOF-74). The proposed solid-phase extraction material possessed a uniform spindle-shaped structure, large surface area (709.60 m2 g-1) and pore volume (0.08 m3 g-1), high metal content (22.57 wt%), which facilitated the interaction between host and guest. As results, the composite displayed outstanding selective recognition and adsorption of IgG, due to synergistic effect of the binding ability of rSPG with the Fc region of IgG, maintained through hydrogen bonding and electrostatic attraction, as well as hydrophobic interaction. The adsorption performance and mechanism of rSPG@Ni-MOF-74 have been fully investigated. Additionally, the rSPG@Ni-MOF-74 composite could effectively separate IgG from serum obtained from healthy humans, with the purity of the separated IgG verified through SDS-PAGE analysis. Furthermore, LC-MS/MS analysis identified a high content of IgG (55.3 %) in the eluate from rSPG@Ni-MOF-74, suggesting the great potential of rSPG@Ni-MOF-74 in IgG separation and enrichment from complex matrix.