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Agglutination of pathogenic microorganisms on the body surface is a significant phenomenon for the prevention of infection. In the present study, we show that an extract of the skin mucus from Japanese flounder (Paralichthys olivaceus) has agglutination activity against the yeast Saccharomyces cerevisiae. We purified this yeast-binding protein, which consists of an approximately 35-kDa homodimer, using affinity chromatography with yeast as a ligand. Multiple internal amino acid sequences of the protein, as determined using liquid chromatography with quadrupole time-of-flight tandem mass spectrometry, mapped to flounder glyceraldehyde 3-phosphate dehydrogenase (GAPDH). An anti-GAPDH antibody inhibited the yeast agglutination activity in the skin mucus extract and stained agglutinated yeast, indicating that flounder GAPDH could agglutinate yeast. The current study suggests that GAPDH, a well-known protein as the sixth enzyme in the glycolytic pathway, is a significant player in mucosal immunity in teleosts., (© 2024 The Societies and John Wiley & Sons Australia, Ltd.)

Recombinant human interleukin-2 (rhIL-2) represents one of the most difficult-to-produce cytokines in E. coli due to its extreme hydrophobicity and high tendency to formation of inclusion bodies. Refolding of rhIL-2 inclusion bodies always represents cumbersome downstream processes and low production efficiency. Herein, we disclosed a fusion strategy for efficiently soluble expression and facile production of rhIL-2 in E. coli Origami B (DE3) host. A two-tandem SUMO fusion partner (His-2SUMO) with a unique SUMO protease cleavage site at C-terminus was devised to fuse with the N-terminus of rhIL-2 and the fusion protein (His-2SUMO-rhIL-2) was almost completely expressed in a soluble from. The fusion partner could be efficiently removed by Ulp1 cleavage and the rhIL-2 was simply produced by a two-step Ni-NTA affinity chromatography with a considerable purity and whole recovery. The eventually obtained rhIL-2 was well-characterized and the results showed that the purified rhIL-2 exhibits a compact and ordered structure. Although the finally obtained rhIL-2 exists in a soluble aggregates form and the aggregation probably has been occurred during expression stage, the soluble rhIL-2 aggregates remain exhibit comparable bioactivity with the commercially available rhIL-2 drug formulation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Calcium-chelating peptides were found in Pacific cod bone, but their binding structure and properties have not been elucidated. Novel calcium-binding peptides were isolated by hydroxyapatite affinity chromatography (HAC), and their binding structure and properties were investigated by isothermal titration calorimetry (ITC), multispectral techniques, and mass spectrometry. Based on multiple purifications, the calcium binding capacity (CBC) of Pacific cod bone peptides (PBPs) was increased from 1.71 ± 0.15 μg/mg to 7.94 ± 1.56 μg/mg. Peptides with a molecular weight of 1-2 kDa are closely correlated with CBC. After binding to calcium, the secondary structure of peptides transitioned from random coil to β-sheet, resulting in a loose and porous microstructure. Hydrogen bonds, electrostatic interaction, and hydrophobic interaction contribute to the formation of peptide‑calcium complexes. The F21 contained 42 peptides, with repeated "GE" motif. Differential structure analysis provides a theoretical basis for the targeted preparation of high CBC peptides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Control interventions recommended by the World Health Organization have successfully resulted in low-intensity schistosomiasis transmission areas. To achieve elimination of transmission, new diagnostic screening tools are needed to overcome less than adequate sensitivity of the currently used Kato-Katz faecal thick smear method. Ideally, in-house serological tests should be avoided due to not having a continuous supply of kits as would be necessary for large population studies. Quality assurance provided by manufacturers and proper performance evaluations are also needed. We evaluated the accuracy of two commercially available serology tests as screening methods for detecting light schistosomiasis infections., Methods: Serum samples were collected in 2015 from individuals living in a low-endemicity locality in northeastern Brazil and deposited in a biorepository. We evaluated immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) and an immunochromatographic test (ICT). The Helmintex method was used to define true-positive samples., Results: Overall sensitivity was close to 90% for both the IgG ELISA and ICT, yet specificity was 28% and 18%, respectively. For the IgM ELISA, the values were estimated to be 55% and 43%, respectively., Conclusions: Poor specificity and positive predictive values prevent these tests from being recommended for screening populations in low-intensity schistosomiasis-endemic areas., (© The Author(s) 2024. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Drug-resistant bacterial infections pose a significant challenge in the field of bacterial disease treatment. Finding new antibacterial pathways and targets to combat drug-resistant bacteria is crucial. The bacterial quorum sensing (QS) system regulates the expression of bacterial virulence factors. Inhibiting bacterial QS and reducing bacterial virulence can achieve antibacterial therapeutic effects, making QS inhibition an effective strategy to control bacterial pathogenicity. This article mainly focused on the PqsA protein in the QS system of Pseudomonas aeruginosa. An affinity chromatography medium was developed using the SpyTag/SpyCatcher heteropeptide bond system. Berberine, which can interact with the PqsA target, was screened from Phellodendron amurense by affinity chromatography. We characterized its structure, verified its inhibitory activity on P. aeruginosa, and preliminarily analyzed its mechanism using molecular docking technology. This method can also be widely applied to the immobilization of various protein targets and the effective screening of active substances., (© 2024 Wiley‐VCH GmbH.)

Borrelia, spirochetes transmitted by ticks, are the etiological agents of numerous multisystemic diseases, such as Lyme borreliosis (LB) and tick-borne relapsing fever (TBRF). This study focuses on two surface proteins from two Borrelia subspecies involved in these diseases: CspZ, expressed by Borrelia burgdorferi sensu stricto (also named BbCRASP-2 for complement regulator-acquiring surface protein 2), and the factor H binding A (FhbA), expressed by Borrelia hermsii. Numerous subspecies of Borrelia, including these latter, are able to evade the immune defenses of a variety of potential vertebrate hosts in a number of ways. In this context, previous data suggested that both surface proteins play a role in the immune evasion of both Borrelia subspecies by interacting with key regulators of the alternative pathway of the human complement system, factor H (FH) and FH-like protein 1 (FHL-1). The recombinant proteins, CspZ and FhbA, were expressed in Escherichia coli and purified by one-step metal-affinity chromatography, with yields of 15 and 20 mg or pure protein for 1 L of cultured bacteria, respectively. The purity was evaluated by SDS-PAGE and HPLC and is close to about 95%. The mass of CspZ and FhbA was checked by mass spectrometry (MS). Proper folding of CspZ and FhbA was confirmed by circular dichroism (CD), and their biological activity, namely their interaction with purified FH from human serum (recombinant FH 15-20  and recombinant FHL-1), was characterized by SPR. Such a study provides the basis for the biochemical characterization of the studied proteins and their biomolecular interactions which is a necessary prerequisite for the development of new approaches to improve the current diagnosis of LB and TBRF. KEY POINTS: • DLS, CD, SEC-MALS, NMR, HPLC, and MS are tools for protein quality assessment • Borrelia spp. possesses immune evasion mechanisms, including human host complement • CspZ and FhbA interact with high affinity (pM to nM) to human FH and rFHL-1., (© 2024. The Author(s).)

Accurate orientations and stable conformations of membrane receptor immobilization are particularly imperative for accurate drug screening and ligand-protein affinity analysis. However, there remain challenges associated with (1) traditional recombination, purification, and immobilization of membrane receptors, which are time-consuming and labor-intensive; (2) the orientations on the stationary phase are not easily controlled. Herein, a novel one-step synthesis and oriented-immobilization membrane-receptor affinity chromatography (oSOMAC) method was developed to realize high-throughput and accurate drug screening targeting specific domains of membrane receptors. We employed Strep-tag II as a noncovalent immobilization tag fused into platelet-derived growth factor receptor β (PDGFRβ) through CFPS, and meanwhile, the Strep-Tactin-modified monolithic columns are prepared in batches. The advantages of oSOMAC are as follows: (1) targeted membrane receptors can be expressed independent of living cell within 1-2 h; (2) orientation of membrane receptors can be flexibly controlled and active sites can expose accurately; and (3) targeted membrane receptors can be synthesized, purified, and orientation-immobilized on monolithic columns in one step. Accordingly, three potential PDGFRβ intracellular domain targeted ligands: tanshinone IIA (Tan IIA), hydroxytanshinone IIA, and dehydrotanshinone IIA were successfully screened out from Salvia miltiorrhiza extract through oSOMAC. Pharmacological experiments and molecular docking further demonstrated that Tan IIA could attenuate hepatic stellate cells activation by targeting the protein kinase domain of PDGFRβ with a K D value of 9.7 μM. Ultimately, the novel oSOMAC method provides an original insight for accurate drug screening and interaction analysis which can be applied in other membrane receptors.

Background: Enamelin is an enamel matrix protein that plays an essential role in the formation of enamel, the most mineralized tissue in the human body. Previous studies using animal models and proteins from natural sources point to a key role of enamelin in promoting mineralization events during enamel formation. However, natural sources of enamelin are scarce and with the current study we therefore aimed to establish a simple microbial production method for recombinant human enamelin to support its use as a mineralization agent., Results: In the study the 32 kDa fragment of human enamelin was successfully expressed in Escherichia coli and could be obtained using immobilized metal ion affinity chromatography purification (IMAC), dialysis, and lyophilization. This workflow resulted in a yield of approximately 10 mg enamelin per liter culture. Optimal conditions for IMAC purification were obtained using Ni 2+ as the metal ion, and when including 30 mM imidazole during binding and washing steps. Furthermore, in vitro mineralization assays demonstrated that the recombinant enamelin could promote calcium phosphate mineralization at a concentration of 0.5 mg/ml., Conclusions: These findings address the scarcity of enamelin by facilitating its accessibility for further investigations into the mechanism of enamel formation and open new avenues for developing enamel-inspired mineralized biomaterials., (© 2024. The Author(s).)

Mitochondrial Membrane Chromatography (MMC) is a bioaffinity chromatography technique developed to study the interaction between target proteins embedded in the mitochondrial membrane and their ligand compounds. However, the MMC stationary phases (MMSP) prepared by chemical immobilization are prone to nonspecific binding in candidate agent screening inevitably. To address these challenges, Twin Strep-Tag/Strep Tactin was employed to establish a specific affinity system in the present study. We prepared a carnitine palmitoyltransferase 1A (CPT1A) MMSP by specifically linking Strep-tactin-modified silica gel with the Twin Strep-Tag on the CPT1A-oriented mitochondrial membrane. This Twin Strep-Tag/Strep Tactin modified CPT1A/MMC method exhibited remarkably better retention behavior, longer stationary phase lifespan, and higher screening specificity compared with previous MMC systems with glutaraldehyde immobilization. We adopted the CPT1A-specific MMC system in screening CPT1A ligands from traditional Chinese medicines, and successfully identified novel candidate ligands: ononin, isoliquiritigenin, and aloe-emodin, from Glycyrrhiza uralensis Fisch and Senna tora (L.) Roxb extracts. Biological assessments illustrated that the compounds screened promote CPT1A enzyme activity without affecting CPT1A protein expression, as well as effectively reduce the lipid droplets and triglyceride levels in the high fat induction HepG2 cells. The results suggest that we have developed an MMC system, which is promising for studying the bioaffinity of mitochondrial membrane proteins to candidate compounds. This system provides a platform for a key step in mitochondrial medicine discovery, especially for bioactive molecule screening from complex herbal extracts.

The polyphenol oxidase (PPO) enzyme, which causes enzymatic browning, has been repeatedly purified from fruit and vegetables by affinity chromatography. In the present research, Sepharose 4B-l-tyrosine-4-amino-2-methylbenzoic acid, a novel affinity gel for the purification of the PPO enzyme with high efficiency, was synthesized. Additionally, Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity gel, known in the literature, was also synthesized, and 9.02, 16.57, and 28.13 purification folds were obtained for the PPO enzymes of potato, mushroom, and eggplant by the reference gel. The PPO enzymes of potato, mushroom, and eggplant were purified 41.17, 64.47, and 56.78-fold from the new 4-amino-2-methylbenzoic acid gel. Following their isolation from the new affinity column, the assessment of PPO enzyme purity involved the utilization of SDS-PAGE. According to the results from SDS-PAGE and native PAGE, the molecular weight of each enzyme was 50 kDa. Then, the inhibition effects of naringin, morin hydrate, esculin hydrate, homovanillic acid, vanillic acid, phloridzin dihydrate, and p-coumaric acid phenolic compounds on purified potato, mushroom, and eggplant PPO enzyme were investigated. Among the tested phenolic compounds, morin hydrate was determined to be the most potent inhibitor on the potato (K i : 0.07 ± 0.03 μM), mushroom (K i : 0.7 ± 0.3 μM), and eggplant (K i : 4.8 ± 1.2 μM) PPO enzymes. The studies found that the weakest inhibitor was homovanillic acid for the potato (K i : 1112 ± 324 μM), mushroom (K i : 567 ± 81 μM), and eggplant (K i : 2016.7 ± 805.6 μM) PPO enzymes. Kinetic assays indicated that morin hydrate was a remarkable inhibitor on PPO., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Given the serious harm caused by dietary intake of diethylstilbestrol (DES), it is urgent to explore rapid and sensitive DES sensing methods. In this work, a photothermal DES immunochromatography sensor based on covalent organic framework (COF) was constructed. The performance of COF in the field of photothermal sensing was systematically investigated for the first time. A donor-acceptor type of COF with a photothermal conversion rate of 51.17 % was synthesized. The logarithm of the DES concentrations-temperature change value standard curve was plotted. The intensity of the photothermal sensing signal was inversely proportional to the sample concentration. The detection limit of the proposed photothermal method (0.24 μg·L -1 ) was 10 times higher than that of visual detection (3 μg·L -1 ). This work not only constructed a novel detection method for DES sensing, but also provided a feasible demonstration for the application of COF in photothermal sensing and expanded the application of their photothermal properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mitochondria perform various metabolic processes that significantly affect cell differentiation, proliferation, signal transduction, and programmed cell death. The disruption of mitochondrial bioenergetic and metabolic functions is closely related to many disorders. The specific isolation and purification of intact, high-purity, and functional mitochondria are central to the understanding of their mechanism of action but remain challenging tasks. In this study, a mitochondrial penetrating peptide (MPP) with the sequence FrFKFrFK(Ac) was used as a mitochondrial recognition motif to construct a peptide-guided affinity separation material. The multiple aromatic phenylalanine (F) residues in this amphiphilic peptide can confer lipophilicity to the mitochondrial membrane, whereas the basic residues (D-arginine and lysine) render the MPP surface positively charged, thereby promoting the binding of negatively charged mitochondria. After the derivatization of the N terminal of MPP with an oligoglycine spacer, the peptide ligands were conjugated to matrix beads (MB) with surface aldehyde functional groups. Peptide functionalization was performed via a condensation reaction between the amino group in the peptide ligand and the aldehyde group on the beads. The generated Schiff bases were reduced, affording stable covalent bonds. The dense and stable functionalization of the beads with the mitochondria-targeting peptides was demonstrated using high performance liquid chromatography (HPLC), zeta potential assay, and scanning electron microscopy (SEM). The immobilization efficiency of the peptide ligands was 1.47 μmol/g, and the surface potential of MB@MPP was 11 mV. MB@MPP was used for the direct isolation of mitochondria after cell homogenization. As observed by SEM, mitochondria with a cross-sectional diameter of 500 nm were efficiently captured on the MB@MPP surface. Because the mitochondrial membrane potential is an important marker of mitochondrial function and the driving force behind the staining of mitochondria with Mito Tracker dyes, the specific binding and separation of fluorescent mitochondria from the cell samples revealed that the proposed MB@MPP-based isolation approach can keep mitochondria intact and retain their functions. Western blot assays were employed to characterize the protein markers of the mitochondria (citrate synthase (CS) and voltage-dependent anion channel protein (VDAC)) and cytoplasmic protein (vinculin), and examine the integrity and purity of the captured mitochondria. The results showed that the lysates released from MB@MPP had high CS and VDAC contents. By contrast, vinculin, which is highly abundant in whole-cell lysates, was barely detected in the lysates from MB@MPP. These results suggest that MB@MPP isolates mitochondria with high affinity, specificity, and antifouling ability by using the targeting peptide as the capture handle. A comparison with a commercial mitochondrial isolation kit demonstrated that MB@MPP can separate mitochondria with higher CS and VDAC abundance and purity. Given the superior separation performance of MB@MPP, the molecular profiles of the isolated mitochondria under stress were subjected to further analysis of their molecular profiles under stress. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established to detect tryptophan (Trp) and riboflavin in the mitochondria. Quantification was performed in multiple-reaction monitoring (MRM) mode. Owing to the high purity of the mitochondria, the Trp and riboflavin contents were determined to be 265 and 0.67 nmol/mg, respectively. The metabolic response of mitochondria to external stimuli was further examined using acadesine, an adenosine 5'-monophosphate (AMP)-activated protein kinase activator with a wide range of metabolic effects, to treat cells. After cell homogenization, MB@MPP was used to separate the mitochondria from the cell samples with and without acadesine treatment, followed by LC-MS/MS analysis. The quantification results demonstrated that acadesine induced a 14% upregulation of Trp content in the mitochondria. By contrast, the riboflavin content decreased to 0.48 nmol/mg, which is 72% of that in untreated mitochondria. The changes in Trp and riboflavin contents could influence their metabolic pathways and, thus, the levels of their metabolites, such as nicotinamide adenine dinucleotide, flavin mononucleotide, and flavin adenine dinucleotide, which are essential coenzymes in mitochondria. Peptide-functionalized affinity microbeads with high affinity and specificity for mitochondria are promising for the efficient isolation of high-quality mitochondria, and offer a useful tool for understanding the complicated functions and dynamics of this unique organelle.

At the end of 2019, an unprecedented outbreak of novel coronavirus pneumonia ravaged the global landscape, inflicting profound harm upon society. Following numerous cycles of transmission, we find ourselves in an epoch where the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coexists alongside influenza viruses (Flu A). Swift and accurate diagnosis of SARS-CoV-2 and Flu A is imperative to stem the spread of these maladies and administer appropriate treatment. Presently, colloidal gold-based lateral flow immunoassays (Au-LFIAs) constructed through electrostatic adsorption are beset by challenges such as diminished sensitivity and feeble binding stability. In this context, we propose the adoption of black polylevodopa nanoparticles (PLDA NPs) featuring abundant carboxyl groups as labeling nanomaterials in LFIA to bolster the stability and sensitivity of SARS-CoV-2 antigens and influenza A virus identifications. The engineered PLDA-LFIAs exhibit the capacity to detect SARS-CoV-2 and Flu A within 30 min, boasting a detection threshold of 5 pg/ml for the SARS-CoV-2 antigen and 0.1 ng/ml for the Flu A H1N1 antigen, thereby underscoring their heightened sensitivity relative to Au-LFIAs. These PLDA-LFIAs hold promise for the early detection of SARS-CoV-2 and Flu A, underscoring the potential of PLDA NPs as a discerning labeling probe to heighten the sensitivity of LFIA across diverse applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Contamination of oilfield chemicals (OFCs) by benzo[a]pyrene (B[a]P) is increasingly becoming a severe environmental security issue. There is an urgent need to develop a rapid and accurate method for B[a]P detection in OFCs. In this study, B[a]P hapten was designed using computer aided molecular design. A high-affinity, specific, and matrix-insensitive monoclonal antibody (mAb) with IC 50 values of 6.77 ng/mL was obtained. Based on this mAb, we developed a rapid gold nanoparticle-based immunochromatographic strip assay (GICA) with double T-line mode for on-site detection of B[a]P in OFCs samples. The GICA exhibited excellent detection performance in OFCs samples with strong acidity, strong alkalinity, and deep color. Under optimal conditions, the proposed method detected B[a]P in OFCs at 0.42-300 mg/kg, and limit of detection was 0.23-1.07 mg/kg. The recovery rate was 88-106% with a coefficient of variation of 1.46-6.35%. Confirmed by natural positive OFCs samples and high-performance liquid chromatography, this GICA is accurate and reliable, with great potential for rapid and cost-effective on-site detection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Leishmaniasis, a group of neglected infectious diseases, encompasses a serious health concern, particularly with visceral leishmaniasis exhibiting potentially fatal outcomes. Nucleoside hydrolase (NH) has a fundamental role in the purine salvage pathway, crucial for Leishmania donovani survival, and presents a promising target for developing new drugs for visceral leishmaniasis treatment. In this study, LdNH was immobilized into fused silica capillaries, resulting in immobilized enzyme reactors (IMERs). The LdNH-IMER activity was monitored on-flow in a multidimensional liquid chromatography system, with the IMER in the first dimension. A C18 analytical column in the second dimension furnished the rapid separation of the substrate (inosine) and product (hypoxanthine), enabling direct enzyme activity monitoring through product quantification. LdNH-IMER exhibited high stability and was characterized by determining the Michaelis-Menten constant. A known inhibitor (1-(β-d-Ribofuranosyl)-4-quinolone derivative) was used as a model to validate the established method in inhibitor recognition. Screening of three additional derivatives of 1-(β-d-Ribofuranosyl)-4-quinolone led to the discovery of novel inhibitors, with compound 2a exhibiting superior inhibitory activity (K i  = 23.37 ± 3.64 µmol/L) compared to the employed model inhibitor. Docking and Molecular Dynamics studies provided crucial insights into inhibitor interactions at the enzyme active site, offering valuable information for developing new LdNH inhibitors. Therefore, this study presents a novel screening assay and contributes to the development of potent LdNH inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Soluble growth stimulation expressed gene 2 (sST2) is a new generation biomarker in the diagnosis and prognosis of heart failure (HF). Here, the sST2-specific aptamers were selected from a random ssDNA library with the full length of 88 nucleotides (nt) via target-immobilized magnetic beads (MB)-based systematic evolution of ligands by exponential enrichment (SELEX) technology. After eight rounds of selection, six aptamers with the most enrichment were selected. Among, the aptamer L1 showed the high-affinity binding to sST2 with the lowest Kd value (77.3 ± 0.05 nM), which was chosen as the optimal aptamer for further molecular docking. Then, the aptamer L1 was used to construct a graphene oxide (GO) - based fluorescence resonance energy transfer (FRET) biosensor for sST2, which exhibits a linear detection range of 0.1-100 μg/ml and a detection limit of 3.7 ng/ml. The aptasensor was applied to detect sST2 in real samples, with a good correlation and agreement with the traditional enzyme-linked immunosorbent assay (ELISA) when quantitative analyzing the sST2 concentration in serum samples from HF patients. The results show that not only an efficient strategy for screening the practicable aptamer, but also a rapid and sensitive detection platform for sST2 were established., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The increasing medical application of virus-like particles (VLPs), notably vaccines and viral vectors, has increased the demand for commercial VLP production. However, VLP manufacturing has not yet reached the efficiency level achieved for recombinant protein therapeutics, especially in downstream processing. This review provides a comprehensive analysis of the challenges associated with affinity chromatography for VLP purification with respect to the diversity and complexity of VLPs and the associated upstream and downstream processes. The use of engineered affinity ligands and matrices for affinity chromatography is first discussed. Although several representative affinity ligands are currently available for VLP purification, most of them have difficulty in balancing ligand universality, ligand selectivity and mild operation conditions. Then, phage display technology and computer-assisted design are discussed as efficient methods for the rapid discovery of high-affinity peptide ligands. Finally, the VLP purification by affinity chromatography is analyzed. The process is significantly influenced by virus size and variation, ligand type and chromatographic mode. To address the updated regulatory requirements and epidemic outbreaks, technical innovations in affinity chromatography and process intensification and standardization in VLP purification should be promoted to achieve rapid process development and highly efficient VLP manufacturing, and emphasis is given to the discovery of universal ligands, applications of gigaporous matrices and platform technology. It is expected that the information in this review can provide a better understanding of the affinity chromatography methods available for VLP purification and offer useful guidance for the development of affinity chromatography for VLP manufacturing in the decades to come., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Breast cancer cell growth is often dependent on the presence of steroidal hormones. The 17β-hydroxysteroid dehydrogenase type 1 isoform (17βHSD1) catalyzes NADPH-dependent conversion of estrone to estradiol, a more potent estrogen, and represents potential drug target for breast cancer treatment.  To provide active enzyme for inhibitor screening, 17βHSD1 is usually expressed in insect or mammalian cells, or isolated from human placenta. In the present study we describe a simple protocol for expression and purification of active human 17βHSD1 from BL21(DE3) Escherichia coli cells. Soluble human 17βHSD1 was expressed using a pET28a(+)-based plasmid, which encodes a hexahistidine tag fused to the N-terminus of the protein, and purified by nickel affinity chromatography. The enzyme activity of purified 17βHSD1 was verified by three methods: thin-layer chromatography, an alkali assay and a spectroscopic assay. These non-radioactive enzyme assays require only standard laboratory equipment, and can be used for screening compounds that modulate 17βHSD1 activity.

Heart failure is a serious medical condition with a poor prognosis. Current treatments can only help manage the symptoms and slow the progression of heart failure. However, there is currently no cure to prevent and reverse cardiac remodeling. Transcription factors are in a central role in various cellular processes, and in the heart, GATA4 and NKX2-5 transcription factors mediate hypertrophic responses and remodeling. We have identified compounds that modulate the synergistic interaction of GATA4 and NKX2-5 and shown that the most promising compound (1, 3i-1000) is cardioprotective in vitro and in vivo. However, direct evidence of its binding site and mechanism of action has not been available. Due to the disordered nature of transcription factors, classical target engagement approaches cannot be utilized. Here, we synthesized a small-molecule ligand-binding pulldown probe of compound 1 to utilize affinity chromatography alongside CETSA, AlphaScreen, and molecular modeling to study ligand binding. These results provide the first evidence of direct physical binding of compound 1 selectively to GATA4. While developing drugs that target transcription factors presents challenges, advances in technologies and knowledge of intrinsically disordered proteins enable the identification of small molecules that can selectively target transcription factors., (© 2024. The Author(s).)

α-Glucosidase plays a direct role in the metabolic pathways of starch and glycogen, any dysfunction in its activity could result in metabolic disease. Concurrently, this enzyme serves as a target for diverse drugs and inhibitors, contributing to the regulation of glucose metabolism in the human body. Here, an integrated analytical method was established to screen inhibitors of α-glucosidase. This step-by-step screening model was accomplished through the biosensing and affinity chromatography techniques. The newly proposed sensing program had a good linear relationship within the enzyme activity range of 0.25 U mL -1 to 1.25 U mL -1 , which can quickly identify active ingredients in complex samples. Then the potential active ingredients can be captured, separated, and identified by an affinity chromatography model. The combination of the two parts was achieved by an immobilized enzyme technology and a microdevice for reaction, and the combination not only ensured efficiency and accuracy for inhibitor screening but also eliminated the occurrence of false positive results in the past. The emodin, with a notable inhibitory effect on α-glucosidase, was successfully screened from five traditional Chinese medicines using this method. The molecular docking results also demonstrated that emodin was well embedded into the active pocket of α-glucosidase. In summary, the strategy provided an efficient method for developing new enzyme inhibitors from natural products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The high-purity phycocyanin has a high commercial value. Most current purification methods of C-phycocyanin involve multiple steps, which are complicated and time-consuming. To solve the problem, this research was studied, and an efficient affinity chromatography purification for C-phycocyanin from Spirulina platensis was developed. Through molecular docking simulation, virtual screening of ligands was performed, and ursolic acid was identified as the specific affinity ligand, which coupled to Affi-Gel 102 gel via 1-ethyl (3-dimethylaminopropyl)-3-carbodiimide, hydrochloride as coupling agent. With this customized and synthesized resin, a high-efficiency one-step purification procedure for C-phycocyanin was developed and optimized, the purity was determined to be 4.53, and the yield was 69 %. This one-step purification protocol provides a new approach for purifying other phycobilin proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

This review summarizes the stepwise strategy and key points for magnetic beads (MBs)-based aptamer selection which is suitable for isolating aptamers against small and large molecules via systematic evolution of ligands by exponential enrichment (SELEX). Particularities, if any, are discussed according to the target size. Examples targeting small molecules (<1000 Da) such as xenobiotics, toxins, pesticides, herbicides, illegal additives, hormones, and large targets such as proteins (biomarkers, pathogens) are discussed and presented in tabular formats. Of special interest are the latest advances in more efficient alternatives, which are based on novel instrumentation, materials or microelectronics, such as fluorescence MBs-SELEX or microfluidic chip system-assisted MBs-SELEX. Limitations and perspectives of MBs-SELEX are also reviewed. Taken together, this review aims to provide practical insights into MBs-SELEX technologies and their ability to screen multiple potential aptamers against targets from small to large molecules., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Immobilized metal ion affinity chromatography (IMAC) adsorbents generally have excellent affinity for histidine-rich proteins. However, the leaching of metal ions from the adsorbent usually affects its adsorption performance, which greatly affects the reusable performance of the adsorbent, resulting in many limitations in practical applications. Herein, a novel IMAC adsorbent, i.e., Cu(II)-loaded polydopamine-coated urchin-like titanate microspheres (Cu-PDA-UTMS), was prepared via metal coordination to make Cu ions uniformly decorate polydopamine-coated titanate microspheres. The as-synthesized microspheres exhibit an urchin-like structure, providing more binding sites for hemoglobin. Cu-PDA-UTMS exhibit favorable selectivity for hemoglobin adsorption and have a desirable adsorption capacity towards hemoglobin up to 2704.6 mg g -1 . Using 0.1% CTAB as eluent, the adsorbed hemoglobin was easily eluted with a recovery rate of 86.8%. In addition, Cu-PDA-UTMS shows good reusability up to six cycles. In the end, the adsorption properties by Cu-PDA-UTMS towards hemoglobin from human blood samples were analyzed by SDS-PAGE. The results showed that Cu-PDA-UTMS are a high-performance IMAC adsorbent for hemoglobin separation, which provides a new method for the effective separation and purification of hemoglobin from complex biological samples.

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). Current vaccine prevention and treatment approaches for PRRS are not adequate, and commercial vaccines do not provide sufficient cross-immune protection. Therefore, establishing a precise, sensitive, simple, and rapid serological diagnostic approach for detecting PRRSV antibodies is crucial. The present study used quantum dot fluorescent microspheres (QDFM) as tracers, covalently linked to the PRRSV N protein, to develop an immunochromatography strip (ICS) for detecting PRRSV antibodies. Monoclonal antibodies against PRRSV nucleocapsid (N) and membrane (M) proteins were both coated on nitrocellulose membranes as control (C) and test (T) lines, respectively. QDFM ICS identified PRRSV antibodies under 10 min with high sensitivity and specificity. The specificity assay revealed no cross-reactivity with the other tested viruses. The sensitivity assay revealed that the minimum detection limit was 1.2 ng/mL when the maximum dilution was 1:2,048, comparable to the sensitivity of enzyme-linked immunosorbent assay (ELISA) kits. Moreover, compared to PRRSV ELISA antibody detection kits, the sensitivity, specificity, and accuracy of QDFM ICS after analyzing 189 clinical samples were 96.7%, 97.9%, and 97.4%, respectively. Notably, the test strips can be stored for up to 6 months at 4 °C and up to 4 months at room temperature (18-25 °C). In conclusion, QDFM ICS offers the advantages of rapid detection time, high specificity and sensitivity, and affordability, indicating its potential for on-site PRRS screening. KEY POINTS: • QDFM ICS is a novel method for on-site and in-lab detection of PRRSV antibodies • Its sensitivity, specificity, and accuracy are on par with commercial ELISA kits • QDFM ICS rapidly identifies PRRSV, aiding the swine industry address the evolving virus., (© 2024. The Author(s).)

Salmonella spp., as one of the foodborne pathogens, is a severe threat to global public health. Rapid screening of salmonella spp. in contaminated food with low infective doses is the key to preventing food poisoning. In this study, a fast visualization method for detecting Salmonella typhimurium (S. typhimurium) was developed based on photonic PCR and AuNPs lateral-flow immunochromatography strip (LFIS). In addition, quantitative detection of target bacteria could be achieved by utilizing the photothermal effect of AuNPs, and the sensitivity could be improved by amplifying the photothermal signal. On the optimized conditions, the developed photonic PCR-LFIS assay was highly sensitive, with a detection limit as low as 19 cfu mL -1 of bacteria in pure culture after laser irradiation, and highly specific, exhibiting no cross-reaction with Salmonella enteritidis, Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus. Notably, S. typhimurium could be detected in pork, egg white, and milk without pre-treatment, with the recovery rates of the three samples between 81 % and 109 %. In conclusion, the photonic PCR-LFIS assay realizes sensitive, simple, and rapid detection of S. typhimurium., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and nicotinamide adenine dinucleotide phosphate (NADPH). Spirulina platensis, which is one of the blue-green algae in the form of spiral rings, belongs to the cyanobacteria class. Spirulina platensis can produce Trx under stress conditions. If it can produce Trx, it also has TrxR activity. Therefore, in this study, the TrxR enzyme was purified for the first time from Spirulina platensis, an algae the most grown and also used as a nutritional supplement in the world. A two-step purification process was used: preparation of the homogenate and 2',5'-ADP sepharose 4B affinity chromatography. The enzyme was purified with a purification fold of 1059.51, a recovery yield of 9.7 %, and a specific activity of 5.77 U/mg protein. The purified TrxR was tested for purity by SDS-PAGE. The molecular weight of its subunit was found to be about 45 kDa. Optimum pH, temperature and ionic strength of the enzyme were pH 7.0, 40 °C and 750 mM in phosphate buffer respectively. The Michaelis constant (K m ) and maximum velocity of enzyme (V max ) values for NADPH and 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) are 5 μM and 2.2 mM, and 0.0033 U/mL and 0.0044 U/mL, respectively. Storage stability of the purified enzyme was determined at several temperatures. The inhibition effects of Ag + , Cu 2+ , Al 3+ and Se 4+ metal ions on the purified TrxR activity were investigated in vitro. While Se 4+ ion increased the enzyme activity, other tested metal ions showed different type of inhibitory effects on the Lineweaver-Burk graphs., Competing Interests: Declaration of competing interest Author declare that they have no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Most proteins are organized in macromolecular assemblies, which represent key functional units regulating and catalyzing most cellular processes. Affinity purification of the protein of interest combined with liquid chromatography coupled to tandem mass spectrometry (AP-MS) represents the method of choice to identify interacting proteins. The composition of complex isoforms concurrently present in the AP sample can, however, not be resolved from a single AP-MS experiment but requires computational inference from multiple time- and resource-intensive reciprocal AP-MS experiments. Here we introduce deep interactome profiling by mass spectrometry (DIP-MS), which combines AP with blue-native-PAGE separation, data-independent acquisition with mass spectrometry and deep-learning-based signal processing to resolve complex isoforms sharing the same bait protein in a single experiment. We applied DIP-MS to probe the organization of the human prefoldin family of complexes, resolving distinct prefoldin holo- and subcomplex variants, complex-complex interactions and complex isoforms with new subunits that were experimentally validated. Our results demonstrate that DIP-MS can reveal proteome modularity at unprecedented depth and resolution., (© 2024. The Author(s).)

Snakes contain three types of phospholipase A 2 (PLA 2 )-inhibitory proteins in their blood, PLIα, β, and γ, which protect them from their own venom, PLA 2 . PLIβ is the snake ortholog of leucine-rich α 2 glycoprotein (LRG). Since autologous cytochrome c (Cyt c ) serves as an endogenous ligand for LRG, in this study, we purified snake LRGs from various snake serum samples using Cyt c affinity chromatography. All purified snake LRGs were found to be dimers linked by disulfide bonds. Laticauda semifasciata and Naja kaouthia LRGs showed no inhibitory activity against L. semifasciata PLA 2 and weak inhibitory activity against Gloydius brevicauda basic PLA 2 . Elaphe climacophora PLIβ had weaker inhibitory activity against G. brevicauda basic PLA 2 than G. brevicauda and Elaphe quadrivirgata PLIs, which are abundant in blood and known to neutralize G. brevicauda basic PLA 2 . Protobothrops flavoviridis LRG showed no inhibitory activity against basic venom PLA 2 , PL-X, or G. brevicauda basic PLA 2 . Binding analysis of P. flavoviridis LRG using surface plasmon resonance showed very strong binding to snake Cyt c , followed by that to horse Cyt c , weak binding to yeast Cyt c , and no binding to P. flavoviridis PL-X or BPI/II. We also deduced the amino acid sequences of L. semifasciata and P. flavoviridis LRG by means of cDNA sequencing and compared them with those of other known sequences of PLIs and LRGs. This study concluded that snake LRG can potentially inhibit basic PLA 2 , but, whether it actually functions as a PLA 2 -inhibitory protein, PLIβ, depends on the snake.

The enzyme α-Galactosidase (α-D-galactoside galactohydrolase [EC 3.2.1.22]) is an exoglycosidase that hydrolyzes the terminal α-galactosyl moieties of glycolipids and glycoproteins. It is ubiquitous in nature and possesses extensive applications in the food, pharma, and biotechnology industries. The present study aimed to purify α-galactosidase from Klebsiella pneumoniae, a bacterium isolated from the human oral cavity. The purification steps involved ammonium sulfate precipitation (70 %), dialysis, ion exchange chromatography using a DEAE-cellulose column, and affinity monolith chromatography. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to determine the molecular weight of the purified enzyme. The kinetic constants, Michaelis constant (Km) and maximal velocity (Vmax), for this enzyme were determined by using p-nitrophenyl-α-D-galactopyranoside as substrate. The results showed that the purification fold, specific activity, and yield were 126.52, 138.58 units/mg, and 21.5 %, respectively. The SDS-PAGE showed that the molecular weight of the purified enzyme was 75 kDa. The optimum pH and temperature of the purified α-galactosidase were detected at pH 6.0 and 50 °C, respectively. The kinetic constants, Michaelis constant (Km) and maximal velocity (Vmax), for this enzyme were 4.6 mM and 769.23 U/ml, respectively. α-galactosidase from Klebsiella pneumoniae was purified and characterized. (SDS-PAGE) analysis showed that the purified enzyme appeared as single band with a molecular weight of 75 kDa., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Conventional techniques for purifying macromolecular conjugates often require complex and costly installments that are inaccessible to most laboratories. In this work, we develop a one-step micropreparative method based on a trilayered polyacrylamide gel electrophoresis (MP-PAGE) setup to purify biological samples, synthetic nanoparticles, as well as biohybrid complexes. We apply this method to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using the conventional crush-and-soak method. MP-PAGE was also able to isolate enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, which is comparable to purities achieved through a more complex two-step purification procedure involving size exclusion and immobilized metal-ion affinity chromatography. This technique was further extended to demonstrate size-dependent separation of a commercial mixture of graphene quantum dots (GQDs) into three different fractions with distinct optical properties. Finally, MP-PAGE was used to isolate DNA-EYFP and DNA-GQD bioconjugates from their reaction mixture of DNA and EYFP and GQD precursors, samples that otherwise could not be effectively purified by conventional chromatography. MP-PAGE thus offers a rapid and versatile means of purifying biological and synthetic nanomaterials without the need for specialized equipment.

Protein hydrolysates from sea cucumber ( Apostichopus japonicus ) gonads are rich in active materials with remarkable angiotensin-converting enzyme (ACE) inhibitory activity. Alcalase was used to hydrolyze sea cucumber gonads, and the hydrolysate was separated by the ultrafiltration membrane to produce a low-molecular-weight peptide component (less than 3 kDa) with good ACE inhibitory activity. The peptide component (less than 3 kDa) was isolated and purified using a combination method of ACE gel affinity chromatography and reverse high-performance liquid chromatography. The purified fractions were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the resulting products were filtered using structure-based virtual screening (SBVS) to obtain 20 peptides. Of those, three noncompetitive inhibitory peptides (DDQIHIF with an IC 50 value of 333.5 μmol·L -1 , HDWWKER with an IC 50 value of 583.6 μmol·L -1 , and THDWWKER with an IC 50 value of 1291.8 μmol·L -1 ) were further investigated based on their favorable pharmacochemical properties and ACE inhibitory activity. Molecular docking studies indicated that the three peptides were entirely enclosed within the ACE protein cavity, improving the overall stability of the complex through interaction forces with the ACE active site. The total free binding energies (ΔG total ) for DDQIHIF, HDWWKER, and THDWWKER were -21.9 Kcal·mol -1 , -71.6 Kcal·mol -1 , and -69.1 Kcal·mol -1 , respectively. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that HDWWKER could significantly decrease the systolic blood pressure (SBP) of SHRs after intravenous administration. The results showed that based on the better antihypertensive activity of the peptide in SHRs, the feasibility of targeted affinity purification and computer-aided drug discovery (CADD) for the efficient screening and preparation of ACE inhibitory peptide was verified, which provided a new idea of modern drug development method for clinical use.

In this study, benzohydroxamic acid molecules were synthesized from methyl 4-amino-2-methoxy, methyl 4-amino-3-nitro, methyl 4-amino-3-methyl, and methyl 4-amino-3-chloro benzoate molecules, and the horseradish peroxidase (HRP) enzyme was purified in one step using the affinity chromatography technique for the first time. The IC 50 and K i values for the 4-amino 3-methyl benzohydroxamic acid molecule were 0.136 and 0.132 ± 0.054 μM, respectively, while the IC 50 and K i values for the 4-amino-3-nitro benzohydroxamic acid molecule were 56.00 and 51.90 ± 9.90 μM, respectively. It was found that the IC 50 and K i values for the 4-amino-3-chloro benzohydroxamic acid molecule were 218.33 and 175.67 ± 43.78 μM, respectively, whereas the IC 50 and K i values for the 4-amino-2-methoxy benzohydroxamic acid molecule were 306.00 and 218.00 ± 68.80 μM, respectively. The HRP enzyme was synthesized from 4-amino-2-methoxy hydroxamic acid column with a 35.97% yield 601.13 times, 4-amino-3-nitro hydroxamic acid column, with a 14.00% yield 404.11 times, 4-amino-3-methyl hydroxamic acid column with an 8.70% yield 394.88 times, and 4-amino-3-chloro hydroxamic acid column with a 4.48% yield 284.85 times. Thus, the HRP enzyme was purified in a single step with hydroxamic acids, and its molecular weight was found to be 44 kDa. The optimum pH was 8.0, the optimum temperature was 15°C, and the optimum ionic strength was 0.4 M for the purified HRP enzyme., (© 2023 International Union of Biochemistry and Molecular Biology, Inc.)

Thioredoxin reductase (TrxR, enzyme code [E.C.] 1.6.4.5) is a widely distributed flavoenzyme that catalyzes nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of thioredoxin and many other physiologically important substrates. Spirulina platensis is a blue-green algae that is often used as a dietary supplement. S. platensis is rich in protein, lipid, polysaccharide, pigment, carotenoid, enzyme, vitamins and many other chemicals and exhibits a variety of pharmacological functions. In the present study, a simple and efficient method to purify TrxR from S. platensis tablets is reported. The extractions were carried out using two different methods: heat denaturation and 2',5'-adenosine diphosphate Sepharose 4B affinity chromatography. The enzyme was purified by 415.04-fold over the crude extract, with a 19% yield, and specific activity of 0.7640 U/mg protein. Optimum pH, temperature and ionic strength of the enzyme activity, as well as the Michaelis constant (K m ) and maximum velocity of enzyme (V max ) values for NADPH and 5,5'-dithiobis(2-nitrobenzoic acid) were determined. Tested metal ions, vitamins, and drugs showed inhibition effects, except Se 4+ ion, cefazolin sodium, teicoplanin, and tobramycin that increased the enzyme activity in vitro. Ag + , Cu 2+ , Mg 2+ , Ni 2+ , Pb 2+ , Zn 2+ , Al 3+ , Cr 3+ , Fe 3+ , and V 4+ ions; vitamin B 3 , vitamin B 6 , vitamin C, and vitamin U and aciclovir, azithromycin, benzyladenine, ceftriaxone sodium, clarithromycin, diclofenac, gibberellic acid, glurenorm, indole-3-butyric acid, ketorolac, metformin, mupirocin, mupirocin calcium, paracetamol, and tenofovir had inhibitory effects on TrxR. Ag + exhibited stronger inhibition than 1-chloro-2,4-dinitrobenzene (a positive control)., (© 2023 International Union of Biochemistry and Molecular Biology, Inc.)

Virus-like particles (VLPs) are nanometric structures composed of structural components of virions, keeping most of the cellular recognition and internalization properties, but are non-infective as they are deprived of their genetic material. VLPs have been a versatile platform for developing vaccines by carrying their own or heterologous antigenic epitopes. Moreover, VLPs can also be used as nanovessels for encapsulating molecules with therapeutic applications, like enzymes, nucleic acids, and drugs. Parvovirus B19 (B19V) VLPs can be self-assembled in vitro from the denatured major viral particle protein VP2 by equilibrium dialysis. Despite its fair productivity, this process is currently a time-consuming task. Affinity chromatography is used as an efficient step for concentration and purification, but it is only sometimes seen as a method that facilitates the oligomerization of proteins. In this research, we report a novel approach for the in vitro assembly of B19V VLPs through the immobilization of the denatured VP2 into an immobilized metal affinity chromatography (IMAC) column, followed by the on-column folding and the final VLP assembly upon protein elution. This method is suitable for the fast production of B19V VLPs. KEY POINTS: • Biotechnological applications for inclusion bodies • Efficient single-step purification and immobilization strategies • Rapid VLP assembly strategy., (© 2024. The Author(s).)

Recently, significant progress has been made in the development of microdevices for point-of-care infectious disease detection. However, most microdevices only allow limited steps, such as DNA amplification on the chip, while sample preparation, such as lysis and DNA extraction, is conducted off the chip using the traditional method. In this study, an all-in-one platform was developed, which incorporated all necessary procedures for nucleic acid detection. Our on-chip DNA extraction method utilized the magnetic bead-based technology on a hybrid channel-digital microfluidics (C-DMF) microdevice. It yielded high recovery rates, varying from 88.43% to 95.83%, with pathogen concentrations of 10 3 -10 6 CFU/mL. In particular, the on-chip method exhibited significantly higher efficacy compared to the traditional off-chip manual method, for the DNA extraction of E. coli and S. aureus , representing Gram-negative and Gram-positive bacteria, respectively, at a sample concentration of 10 3 CFU/mL. To address the need for rapid and accessible diagnostics, colorimetric LAMP amplification was integrated into the proposed microdevice. The results were visually detectable with the naked eye, making it user-friendly for non-specialists. In addition, this platform demonstrated impressive sensitivity in simultaneously detecting common foodborne pathogens in spiked meat samples, achieving the LOD of 10 2 -10 3 CFU/mL. The entire process, from sampling to result, was fully automated and only required approximately 60 min, offering promising applicability in resource-limited and on-site testing scenarios.

Background: Protease 3C (3Cpro) is the only protease encoded in the human hepatitis A virus genome and is considered as a potential target for antiviral drugs due to its critical role in the viral life cycle. Additionally, 3Cpro has been identified as a potent inducer of ferroptosis, a newly described type of cell death. Therefore, studying the molecular mechanism of 3Cpro functioning can provide new insights into viral-host interaction and the biological role of ferroptosis. However, such studies require a reliable technique for producing the functionally active recombinant enzyme., Objective: Here, we expressed different modified forms of 3Cpro with a hexahistidine tag on the N- or C-terminus to investigate the applicability of immobilized metal Ion affinity chromatography (IMAC) for producing 3Cpro., Methods: We expressed the proteins in Escherichia coli and purified them using IMAC, followed by gel permeation chromatography. The enzymatic activity of the produced proteins was assayed using a specific chromogenic substrate., Results: Our findings showed that the introduction and position of the hexahistidine tag did not affect the activity of the enzyme. However, the yield of the target protein was highest for the variant with seven C-terminal residues replaced by a hexahistidine sequence., Conclusion: We demonstrated the applicability of our approach for producing recombinant, enzymatically active 3Cpro., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Adeno-associated viruses (AAVs) have acquired a central role in modern medicine as delivery agents for gene therapies targeting rare diseases. While new AAVs with improved tissue targeting, potency, and safety are being introduced, their biomanufacturing technology is lagging. In particular, the AAV purification pipeline hinges on protein ligands for the affinity-based capture step. While featuring excellent AAV binding capacity and selectivity, these ligands require strong acid (pH <3) elution conditions, which can compromise the product's activity and stability. Additionally, their high cost and limited lifetime has a significant impact on the price tag of AAV-based therapies. Seeking to introduce a more robust and affordable affinity technology, this study introduces a cohort of peptide ligands that (i) mimic the biorecognition activity of the AAV receptor (AAVR) and anti-AAV antibody A20, (ii) enable product elution under near-physiological conditions (pH 6.0), and (iii) grant extended reusability by withstanding multiple regenerations. A20-mimetic CYIHFSGYTNYNPSLKSC and AAVR-mimetic CVIDGSQSTDDDKIC demonstrated excellent capture of serotypes belonging to distinct clones/clades - namely, AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. This corroborates the in silico models documenting their ability to target regions of the viral capsid that are conserved across all serotypes. CVIDGSQSTDDDKIC-Toyopearl resin features binding capacity (≈10 14 vp mL -1 ) and product yields (≈60%-80%) on par with commercial adsorbents, and purifies AAV2 from HEK293 and Sf9 cell lysates with high recovery (up to 78%), reduction of host cell proteins (up to 700-fold), and high transduction activity (up to 65%)., (© 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Background: DDX3 is a protein with RNA helicase activity that is involved in a variety of biological processes, and it is an important protein target for the development of broad-spectrum antiviral drugs, multiple cancers and chronic inflammation., Objectives: The objective of this study is to establish a simple and efficient method to express and purify DDX3 protein in E. coli, and the recombinant DDX3 should maintain helicase activity for further tailor-made screening and biochemical function validation., Methods: DDX3 cDNA was simultaneously cloned into pET28a-TEV and pNIC28-Bsa4 vectors and transfected into E. coli BL21 (DE3) to compare one suitable prokaryotic expression system. The 6×His-tag was fused to the C-terminus of DDX3 to form a His-tagging DDX3 fusion protein for subsequent purification. Protein dissolution buffer and purification washing conditions were optimized. The His-tagged DDX3 protein would bind with the Ni-NTA agarose by chelation and collected by affinity purification. The 6×His-tag fused with N-terminal DDX3 was eliminated from DDX3 by TEV digestion. A fine purification of DDX3 was performed by gel filtration chromatography., Results: The recombinant plasmid pNIC28-DDX3, which contained a 6×His-tag and one TEV cleavage site at the N terminal of DDX3 sequence, was constructed for DDX3 prokaryotic expression and affinity purification based on considering the good solubility of the recombinant His-tagging DDX3, especially under 0.5 mM IPTG incubation at 18°C for 18 h to obtain more soluble DDX3 protein. Finally, the exogenous recombinant DDX3 protein was obtained with more than 95% purity by affinity purification on the Ni-NTA column and removal of miscellaneous through gel filtration chromatography. The finely-purified DDX3 still retained its ATPase activity., Conclusion: A prokaryotic expression pNIC28-DDX3 system is constructed for efficient expression and affinity purification of bioactive DDX3 protein in E. coli BL21(DE3), which provides an important high-throughput screening and validation of drugs targeting DDX3., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Antibodies are versatile biological molecules with widespread applications in research and medicine. This protocol outlines the generation of monoclonal IgG antibodies from Chinese hamster ovary cells. It includes steps for cell maintenance, transient transfection, and antibody purification via protein A affinity chromatography. The methods described are intended for the production of milligram amounts of protein but can be adapted for most small- to mid-scale applications., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This chapter presents a method for the heterologous expression and purification of human ALA synthase from Escherichia coli. Mature ALAS is produced with an N-terminal hexahistidine affinity tag followed by a SUMO fusion tag for solubility and ease of purification. The plasmid is introduced into competent E. coli cells, and robust protein expression is induced with IPTG. The ALAS cofactor, pyridoxal 5'-phosphate, is inserted during protein production to yield an active enzyme upon purification. After cell lysis, the tagged ALAS protein is isolated via a multistep purification that involves an initial nickel-affinity step, affinity tag cleavage and removal, and a final size exclusion chromatography polishing step. Importantly, this protocol is amenable to various ALAS truncations and mutations, opening the door to understanding ALAS biology and its intersections with iron utilization across several organisms., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

High throughput allergen characterization is often based on phage display technique which is limited by the constraints of a prokaryotic expression system such as potential loss of conformational epitopes and lack of post-translational modifications. Replacing the phage display platform with a yeast surface display system could accelerate the immunological characterization of complex structured allergens. Yeast surface display is a powerful technique that allows faster immunochemical characterization of allergens in situ without the need for protein purification. Yeast surface display offers an alternative that could lead to the improvement of standard immunodiagnostic and immunotherapeutic approaches. In this chapter, we describe a protocol on yeast surface display for the characterization of plant-derived food allergens using actinidin (Act d 1), a major kiwifruit allergen, as a model system., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The discovery of protein-protein interaction networks can lead to the unveiling of protein complex(es) forming cellular machinerie(s) or reveal component proteins of a specific cellular pathway. Deciphering protein-protein interaction networks therefore contributes to a deeper understanding of how cells function. Here we describe the protocol to perform tandem affinity purification (TAP) in bacteria, which enables the identification of the partners of a bait protein under native conditions. This method consists in two sequential steps of affinity purification using two different tags. For that purpose, the bait protein is translationally fused to the TAP tag, which consists of a calmodulin-binding peptide (CBP) and two immunoglobulin G (IgG)-binding domains of Staphylococcus aureus protein A (ProtA) that are separated by the tobacco etch virus (TEV) protease cleavage site. After the first round of purification based on the binding of ProtA to IgG-coated beads, TEV protease cleavage releases CBP-tagged bait protein along with its partners for a second round of purification on calmodulin affinity resin and leaves behind protein contaminants bound to IgG. Creating the TAP-tag translational fusion at the chromosomal locus allows detection of protein interactions occurring in physiological conditions., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)