Protein Conjugates Research Articles

Ultrasonic assisted preparation of covalent bonding pea protein and polyphenol conjugate in emulsion delivery system

Protein modification can improve the bioavailability of polyphenols, while polyphenols can also improve the functional properties of pea protein. Four representative polyphenols (gallic acid, ferulic acid, catechin and kaempferol) were covalently combined with pea protein by ultrasonic assisted grafting (free radical grafting, laccase method and alkali treatment), and the structure and functional properties of the conjugated compounds were characterized. The interaction was evaluated by surface hydrophobicity, free sulfhydryl content determination and SDS-PAGE. Fluorescence spectra and Fourier transform infrared spectra revealed that polyphenol binding reduced the α-helix content of pea protein and changed its structure. In addition, the antioxidant activity of the conjugate was significantly improved compared to natural pea protein (DPPH by 7.5 to 20.2 times, ABTS+ by 2.8 to 4.3 times, FRAP by 1.4 to 4.1 times). At the same time, the introduction of polyphenols also optimized the thermal stability of pea protein, and increased the denaturation temperature of pea protein (113.1 °C) by 0.19–6.63 °C. The emulsifying properties were also enhanced by covalent modification, and the emulsifying properties of the ultrasound-assisted conjugates were better. In summary, the pea protein-polyphenol conjugate provides a new solution for improving the bioavailability of polyphenols and preparing polyphenol delivery systems for complex emulsions.

Digital insights into Pseudomonas aeruginosa PBH03: in-silico analysis for genomic toolbox and unraveling cues for heavy metal bioremediation.

The genomes of publicly available electroactive Pseudomonas aeruginosa strains are currently limited to in-silico analyses. This study analyzed the electroactive Pseudomonas aeruginosa PBH03 genome using comparative in-silico studies for biotechnological applications. Comparative in-silico and experimental analyses were conducted to identify the novel traits of P. aeruginosa PBH03 by genome sequencing. The publicly available genomes of Pseudomonas aeruginosa strains (PA01, PA14, and KRP1) were used for a comparative in-silico study with PBH03. Genome assembly, annotation, phylogenetic analysis, metabolic reconstruction, and comparative functional genes analysis were conducted using bioinformatics tools. The experimental analyses were conducted to validate the heavy metal resistance (Hg and Cu), salinity tolerance levels of PBH03, and acetate assimilation under microaerobic conditions. Computational analysis showed that the PBH03 genome had a size of 6.8Mb base pairs with a GC content of 65.7%. Whole genome annotation identified the unique genes absent in the previously sequenced Pseudomonas aeruginosa genomes. These genes were associated with resistance to heavy metals, such as Cu, Hg, As, and a Co-Zn-Cd efflux system. In addition, clustered, regularly interspaced short palindromic repeats, transposable elements, and conjugative transfer proteins were observed in the clustering-based systems. The strain exhibited resistance to Hg (150mg/L) and Cu (500mg/L) and showed growth at salinity levels of 40g/L (typical sea/ocean levels). PBH03 could consume acetate up to 110mM. Integrating in-silico and experimental data highlights the intriguing adaptive genomic qualities of PBH03, making it a promising candidate for various biotechnological applications.

Dual-Performing Vinyltetrazine for Rapid, Selective Bioconjugation and Functionalization of Cysteine Proteins.

Although methods for Cys-specific bioconjugation and functionalization of proteins have been developed and widely utilized in biomolecule engineering and therapeutic development, reagents for this purpose are generally designed to accomplish bioconjugation only. Consequently, additional clickable groups must be attached to these reagents to accomplish functionalization. Herein, we describe a new, simple, dual-performing bioconjugation-functionalization reagent, VMeTz, which possesses an electron-withdrawing tetrazine (Tz) substituted vinyl (V) moiety to serve as both a Michael receptor for selective conjugation with Cys and a site for click with TCO derivatives to introduce functionality. Critically, VMeTz contains a methyl group that prevents the formation of multiple Tz-containing Cys-adducts. Reactions of VMeTz with Cys-containing peptides and proteins both in vitro and in live cells produce single stable Michael adducts with high selectivity. Moreover, the Cys-VMeTz peptide and protein conjugates undergo facile click reactions with TCO-functionalized reagents for labeling and protein profiling. Furthermore, VMeTz selectively activates and delivers the TCO-caged toxic substances Dox and PROTAC ARV-771 to cancer cells to produce therapeutic effects that are comparable to those of the parent drugs. Collectively, the studies demonstrate that VMeTz is a useful reagent for therapeutically significant Cys-specific protein bioconjugation and functionalization.

Conjugation Strategies for Low Solubility Proteins to Single‐Walled Carbon Nanotubes as a Sensitive Fluorescent Assay to Protease Activity

AbstractSingle‐walled carbon nanotube (SWCNT)‐based optical biosensors are shown to detect hydrolase activity directly on target substrates. This study presents a metastable protein conjugation approach to immobilize hydrophobic proteins and enhance sensitivity of protease detection. The method combines covalent conjugation of substrate proteins via carbodiimide chemistry with non‐covalent polymer wrapping of SWCNT, carboxymethyl cellulose (CMC). The formation of protein‐SWCNT complexes as a result of multi‐site conjugation between the proteins and carboxyl groups, enabled iterative pelleting, washing, and resuspension steps to be applied to the probes which allowed for the removal of unbound proteins and residual materials, enhancing the sensor's sensitivity by approximately threefold, reaching a limit of detection (LOD) of 6.4 ng ml−1 in a 5 minute reaction. This immobilization approach is applied to extracellular matrix (ECM) proteins such as gelatin and collagen to detect ECM degrading enzyme activity. ECM degrading enzymes caused a fluorescent intensity decrease of the SWCNT probes, enabling quantification of enzyme concentration between the range 160 ng ml−1 to 100 µg ml−1 within 5 minutes of reaction. This hybrid approach provides a rapid and sensitive platform for detecting extracellular degrading enzymes with potential applications in cancer diagnosis and prognosis, wound healing, high‐throughput screening for enzyme inhibitors, and drug discovery.

The utilization of an ultrasonic mung bean protein-starch conjugate as a fat substitute in whipping cream.

Amidst the rising trend of healthy eating, there is a surge in demand for low-fat food options. Within the realm of fat substitutes, modified proteins have shown the most effective ability to replace fat due to their nutritional attributes and functional properties. This study focused on the development of a fat substitute for low-fat whipping cream using the conjugate of ultrasonic mung bean protein and mung bean starch. Our findings revealed that the emulsifying properties and solubility of the conjugates were significantly superior to those of mung bean protein alone (P < 0.05). This enhancement was attributed to a smaller particle size, depolymerization of protein molecules and increased total sulfhydryl content, especially the conjugate formed by 60 min ultrasonic mung bean protein and mung bean starch (UMBP60+MS). Incorporating UMBP60+MS as a fat substitute at a 10% ratio in the formulation of low-fat whipping cream resulted in a product with enhanced apparent viscosity, superior environmental stability, and commendable sensory characteristics. Moreover, the fat digestion rate was significantly reduced by 13.5% with the 10% substitution. This 10% substitution also endowed the whipping cream with the most desirable β'-type crystal morphology and the most stable three-dimensional network structure. An intimate encapsulation of fat globules by the conjugate was observed using cryogenic scanning electron microscopy. The UMBP60+MS conjugate emerged as an effective fat substitute in whipping cream, providing significant contributions to addressing health concerns in the food industry. © 2024 Society of Chemical Industry.

Anticoagulant Activity of the Polysaccharide Fromgonad of Abalone Haliotis discus hannai Ino: The Role of Conjugate Protein.

Few studies are concerned with the effect of the conjugat protein on the bioactivities of the abalone gonad polysaccharide (AGP). In this study, a series of treatments, including raw material (female and male) defatting, extraction temperature (25-121 °C), proteolysis, ultrafiltration, and ethanol precipitation, was conducted to investigate the role of the conjugate protein on AGP anticoagulant activity. All AGP extracts significantly prolonged activated partial thromboplastin time (APTT) and thrombin time (TT). The strongest was observed in the female AGPs prepared at 50 and 121 °C. The most active is located at 30-300 kDa by ultrafiltration. After being exposed to neutral protease, quick shortening of APTT and TT was found in all AGPs. Further ethanol precipitating of found the longest APTT in the sediment, which contains most polysaccharides and proteins. Defatting lowered the activity of female AGP but increased that of males. Proteolysis also significantly weakened the clotting factor inhibition effect of the 50 °C female AGP, but heating seemed not affect the effect. Five fractions were obtained after the 50 °C female AGP was subjected to ion exchange column. Fraction V, with the highest protein and medium polysaccharide content, showed the strongest anticoagulant effect and was also much higher than AGSP, which was obtained by multi-step proteolysis. The findings supported positive effect of the conjugate protein in AGP anticoagulant activity.

Mechanistic basis for protein conjugation in a diverged bacterial ubiquitination pathway.

Ubiquitination is a fundamental and highly conserved protein post-translational modification pathway, in which ubiquitin or a ubiquitin-like protein (Ubl) is typically conjugated to a lysine side chain of a target protein. Ubiquitination is a multistep process initiated by adenylation of the Ubl C-terminus, followed by sequential formation of 2-3 Ubl~cysteine thioester intermediates with E1, E2, and E3 proteins before formation of the final Ubl-lysine isopeptide bond1. Ubiquitination is conserved across eukaryotes, and recent work has also revealed at least two related bacterial pathways that perform protein conjugation in the context of antiphage immunity2-5. Bioinformatics analysis has hinted at the existence of additional, as-yet uncharacterized, pathways in bacteria that could perform protein conjugation using ubiquitination-like machinery6-8. Here we describe the architecture and biochemical mechanisms of Bub (bacterial ubiquitination-like) pathways, revealing strong structural parallels along with striking mechanistic differences when compared to eukaryotic ubiquitination pathways. We show that Bub operons encode functional E1, E2, and Ubl proteins that are related to their eukaryotic counterparts but function entirely through oxyester, rather than thioester, intermediates. We also identify a novel family of serine proteases in Bub operons with a conserved serine-histidine catalytic dyad. The genomic context of Bub operons suggests that, like other bacterial ubiquitination-related pathways, they also function in antiphage immunity. Overall, our results reveal a new family of bacterial ubiquitination-related pathways with unprecedented biochemical mechanisms in both protein conjugation and deconjugation.

Plasmodium berghei liver stage parasites exploit host GABARAP proteins for TFEB activation

Plasmodium, the causative agent of malaria, infects hepatocytes prior to establishing a symptomatic blood stage infection. During this liver stage development, parasites reside in a parasitophorous vacuole (PV), whose membrane acts as the critical interface between the parasite and the host cell. It is well-established that host cell autophagy-related processes significantly impact the development of Plasmodium liver stages. Expression of genes related to autophagy and lysosomal biogenesis is orchestrated by transcription factor EB (TFEB). In this study, we explored the activation of host cell TFEB in Plasmodium berghei-infected cells during the liver stage of the parasite. Our results unveiled a critical role of proteins belonging to the Gamma-aminobutyric acid receptor-associated protein subfamily (GABARAP) of ATG8 proteins (GABARAP/L1/L2 and LC3A/B/C) in recruiting the TFEB-blocking FLCN-FNIP (Folliculin-Folliculin-interacting protein) complex to the PVM. Remarkably, the sequestration of FLCN-FNIP resulted in a robust activation of TFEB, reliant on conjugation of ATG8 proteins to single membranes (CASM) and GABARAP proteins. Our findings provide novel mechanistic insights into host cell signaling occurring at the PVM, shedding light on the complex interplay between Plasmodium parasites and the host cell during the liver stage of infection.

One‐Step Maleimide‐Based Dual Functionalization of Protein N‐Termini

Maleimide derivatives are privileged reagents for chemically modifying proteins through the Michael addition reaction with cysteine due to their selectivity, operational simplicity, and commercial availability. However, since accessible free cysteine is rarely found in natural proteins, it is highly desirable to find alternative targets to enable direct bioconjugation of proteins with maleimides. In this study, we have developed an operationally simple and straightforward method for the N‐terminal modification of proteins without the need for mutagenesis via a copper(II)‐mediated [3+2] cycloaddition reaction with maleimides and 2‐pyridinecarboxaldehyde (2‐PCA) derivatives under non‐denaturing conditions at pH 6 and 37 °C in aqueous media. Our method utilizes commercially available maleimides to attach diverse functionalities to various N‐terminal amino acids. We demonstrate the preparation of a ternary protein complex cross‐linked at the N‐termini and dually modified trastuzumab equipped with monomethyl auristatin E (MMAE), a cytotoxic agent, and a Cy5 fluorophore (MMAE–Cy5–trastuzumab). MMAE–Cy5–trastuzumab retained human epidermal growth factor receptor 2 (HER2) recognition activity and exerted cytotoxicity against HER2‐positive cells. Furthermore, MMAE–Cy5–trastuzumab allowed successful visualization of HER2‐positive cancer cells in mouse tumors. This straightforward method will expand the accessibility of protein conjugates with well‐defined structures in a wide range of research fields.

One-Step Maleimide-Based Dual Functionalization of Protein N-Termini.

Maleimide derivatives are privileged reagents for chemically modifying proteins through the Michael addition reaction with cysteine due to their selectivity, operational simplicity, and commercial availability. However, since accessible free cysteine is rarely found in natural proteins, it is highly desirable to find alternative targets to enable direct bioconjugation of proteins with maleimides. In this study, we have developed an operationally simple and straightforward method for the N-terminal modification of proteins without the need for mutagenesis via a copper(II)-mediated [3+2] cycloaddition reaction with maleimides and 2-pyridinecarboxaldehyde (2-PCA) derivatives under non-denaturing conditions at pH 6 and 37 °C in aqueous media. Our method utilizes commercially available maleimides to attach diverse functionalities to various N-terminal amino acids. We demonstrate the preparation of a ternary protein complex cross-linked at the N-termini and dually modified trastuzumab equipped with monomethyl auristatin E (MMAE), a cytotoxic agent, and a Cy5 fluorophore (MMAE-Cy5-trastuzumab). MMAE-Cy5-trastuzumab retained human epidermal growth factor receptor 2 (HER2) recognition activity and exerted cytotoxicity against HER2-positive cells. Furthermore, MMAE-Cy5-trastuzumab allowed successful visualization of HER2-positive cancer cells in mouse tumors. This straightforward method will expand the accessibility of protein conjugates with well-defined structures in a wide range of research fields.

Design of Thermo-Responsive Pervaporation Membrane Based on Hyperbranched Polyglycerols and Elastin-like Protein Conjugates.

This paper reports the development of a highly crosslinked hyper-branched polyglycerol (HPG) polymer bound to elastin-like proteins (ELPs) to create a membrane that undergoes a distinct closed-to-open permeation transition at 32 °C. The crosslinked HPG forms a robust, mesoporous structure (150-300 nm pores), suitable for selective filtration. The membranes were characterized by FTIR, UV-visible spectroscopy, SEM, and AFM, revealing their structural and morphological properties. Incorporating a synthetic polypeptide introduced thermo-responsive behavior, with the membrane transitioning from impermeable to permeable above the lower critical solution temperature (LCST) of 32 °C. Permeation studies using crystal violet (CV) demonstrated selective transport, where CV permeated only above 32 °C, while water permeated at all temperatures. This hybrid HPG-ELP membrane system, acting as a molecular switch, offers potential for applications in drug delivery, bioseparations, and smart filtration systems, where permeability can be controlled by temperature.

Automated radiofluorination of HER2 single domain antibody: the road towards the clinical translation of [18F]FB-HER2 sdAb

BackgroundWith the next generation of Human Epidermal Growth Factor Receptor 2 (HER2) -targeting therapies, such as antibody–drug conjugates, showing benefit in “HER2 low” and even “HER2 ultralow” patients, the need for novel methods to quantify HER2 expression accurately becomes even more important for clinical decision making. A HER2 PET/CT imaging assessment could evaluate HER2 positive disease locations while improving patient care, reducing the need for invasive biopsies. A single-domain antibody (sdAb)-based PET tracer could combine the high specificity of sdAbs with short-lived radionuclides such as fluorine-18 (18F) and gallium-68 (68Ga). SdAb-based PET tracers have clinically been used via a 68Ga-chelator approach. However, the distribution of 68Ga-labelled pharmaceuticals to peripheral PET centres is more challenging to organize due to the short half-life of 68Ga, most certainly when the available activity is limited by a generator. Cyclotron produced 68Ga has removed this limitation. Distribution of 18F-labelled pharmaceuticals remains less challenging due to its slightly longer half-life, and radiofluorination of sdAbs via N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) has shown to be a promising strategy for developing sdAb-based PET tracers. Although [18F]SFB automation has been reported, automating protein conjugation proves challenging. Herein we report the fully automated, cartridge-based production of [18F]FB-HER2 sdAb on a single synthesis module.Results[18F]FB-HER2 sdAb (> 6 GBq) was obtained after a fully automated production (95 min), with a RCP > 95%, apparent molar activity > 20 GBq/µmol and decay-corrected radiochemical yield (RCY d.c.) of 14 ± 2% (n = 4). Further upscaling amounted to production batches of 16 GBq with an apparent molar activity > 40 GBq/µmol and RCY d.c. of 8 ± 1% (n = 4). Ex vivo biodistribution and PET imaging showed specific HER2-positive tumour targeting and low kidney retention.ConclusionThe [18F]FB-HER2 sdAb tracer was produced with clinically relevant activities using a fully automated production method. The automated production method was designed to ease the translation to the clinic and has the potential to be used not only in mono-centre but also multi-centre clinical trials with one central production site. [18F]FB-HER2 sdAb showed a favourable biodistribution pattern and could be a valuable alternative to 68Ga-labelled sdAb-based PET tracers in the clinic.

Covalent Attachment of Functional Proteins to Microfiber Surfaces via a General Strategy for Site-Selective Tetrazine Ligation.

Surface modification of materials with proteins has various biological applications, and hence the methodology for surface modification needs to accommodate a wide range of proteins that differ in structure, size, and function. Presented here is a methodology that uses the Affinity Bioorthogonal Chemistry (ABC) tag, 3-(2-pyridyl)-6-methyltetrazine (PyTz), for the site-selective modification and purification of proteins and subsequent attachment of the protein to trans-cyclooctene (TCO)-functionalized hydrogel microfibers. This method of surface modification is shown to maintain the functionality of the protein after conjugation with proteins of varying size and functionalities, namely, HaloTag, NanoLuc luciferase (NanoLuc), and fibronectin type III domains 9-10 (FNIII 9-10). The method also supports surface modification with multiple proteins, which is shown by the simultaneous conjugation of HaloTag and NanoLuc on the microfiber surface. The ability to control the relative concentrations of multiple proteins presented on the surface is shown with the use of HaloTag and superfolder GFP (sfGFP). This application of the ABC-tagging methodology expands on existing surface modification methods and provides flexibility in the site-selective protein conjugation methods used along with the rapid kinetics of tetrazine ligation.

Designing ferulic acid-functionalized dual protein conjugates: Process-induced changes, structural interplays, and sustainable calcium fortification

This study investigates the structural changes and covalent interactions of rice glutelin and egg white proteins with ferulic acid, considering the effects of germination and whisking times. Using ternary phase diagrams, we analyzed the antioxidant properties and in vitro digestive behaviors of these protein-ferulic acid conjugates. Increased germination time enhanced the conjugate potential of rice glutelin, while prolonged whisking time increased the molecular flexibility of egg white proteins. The optimal ratio of rice glutelin to egg white proteins (6:4) resulted in higher surface hydrophobicity. In vitro digestion showed that a higher proportion of rice glutelin aids gastric digestion, while more egg white protein supports intestinal hydrolysis. Extended germination time reduced particle size and antioxidant capacities during digestion but negatively impacted calcium loading from eggshells. These findings highlight the potential of tailored protein-ferulic acid conjugates for improved functional properties and food applications.

Maillard reaction-based conjugation of pea protein and prebiotic (polydextrose): optimization, characterization, and functional properties enhancement

There is a growing demand for plant-based protein ingredients with improved functionality for use in diverse food and nutraceutical applications. In line with this, the current study aimed to investigate the effect of plant protein-prebiotic (polydextrose) conjugation on the techno-functional properties (emulsification, solubility, fat absorption and foaming) of pea proteins through wet heating Maillard reaction. Pea protein (PeP) was conjugated with polydextrose by incubating the mixture at various process conditions (pea protein: polydextrose mass ratios, temperature, and time). Response surface methodology coupled with Box–Behnken design was used to optimize multiple responses, including conjugation efficiency (CE), emulsifying activity (EAI), and foaming capacity (FC). The pea protein conjugate (optimized value) showed improved solubility throughout a wide pH (2–10) range and higher emulsification activity than pea protein alone. The development of conjugates (PeC) was validated through ultraviolet–visible spectroscopy, FTIR, and o-Phthaldialdehyde (OPA) assay. Browning index, FT-IR spectra, thermal properties, and scanning electron microscopy (SEM) micrographs were analyzed for the conjugate (PeC) obtained at optimized values. The FTIR spectra of the conjugates showed new peaks at 3100–3480 cm−1 and 1,000–1,166 cm−1 indicating conjugation. The Maillard conjugation increased the proportion of β-turn, random coil, accompanied by a decrease in α-helix, and β-sheet. These conformational changes were associated to the improved techno-functional properties of the pea protein upon conjugation, offering potential applications in the formulation of plant-based foods and beverages.

Glycation of sunnhemp protein with dextran via dry heating: Thermal, micro-structural characterization, and amino acid profiling.

This study aims to obtain sunnhemp protein isolate (SHPI) and dextran conjugates by dry heating method of Maillard conjugation. The effects of different incubation time (0, 1, 3, 5, 7, and 9 days) on the molecular flexibility, available lysine content, antioxidant properties, molecular structure, and thermal and micro-structural properties of conjugates were compared with SHPI (no conjugation) at 60°C and 79% relative humidity. The results indicated the formation of SHPI-dextran conjugates as confirmed by the change in molecular flexibility, lysine content, antioxidant activities, color, and water activity values. The molecular structure revealed the confirmation of covalent bonding between SHPI and dextran. Differential scanning calorimetry and thermo-gravimetric analysis results exhibited improvement in the thermal stability of SHPI when conjugated with dextran. The microstructural characterization showed that Maillard conjugation changed the surface structure of SHPI. The analysis of amino acid composition displayed that lysine, arginine, and phenylalanine were the dominant Maillard reaction sites of SHPI and dextran. Among all the conjugated samples, 5 days of incubation time was selected as an optimum condition for the development of SHPI-dextran conjugates on the basis of the aforementioned characterization. Overall, it was concluded that Maillard conjugation of sunnhemp protein with dextran via dry-heating technique could modify and improve its various attributes. PRACTICAL APPLICATION: The conjugation of plant proteins with polysaccharide through the Maillard reaction under dry heating conditions represents a natural and green technique for improving the techno-functional properties of proteins. The study has the potential to establish framework for the utilization of Sunnhemp protein isolate-dextran conjugates. This approach offers the potential for cost-effective production of emulsifiers and development of effective encapsulating matrices. The investigation expands on an underutilized plant protein source facilitating an alternative to animal-based proteins and contributing to the development of a sustainable circular bioeconomy.

Analysis of Acinetobacter P-type type IV secretion system-encoding plasmid diversity uncovers extensive secretion system conservation and diverse antibiotic resistance determinants.

Acinetobacter baumannii is globally recognized as a multi-drug-resistant pathogen of critical concern due to its capacity for horizontal gene transfer and resistance to antibiotics. Phylogenetically diverse Acinetobacter species mediate human infection, including many considered as important emerging pathogens. While globally recognized as a pathogen of concern, pathogenesis mechanisms are poorly understood. P-type type IV secretion systems (T4SSs) represent important drivers of pathogen evolution, responsible for horizontal gene transfer and secretion of proteins that mediate host-pathogen interactions, contributing to pathogen survival, antibiotic resistance, virulence, and biofilm formation. Genes encoding a P-type T4SS were previously identified on plasmids harboring the carbapenemase gene blaNDM-1 in several clinically problematic Acinetobacter; however, their prevalence among the genus, geographical distribution, the conservation of T4SS proteins, and full capacity for resistance genes remain unclear. Using systematic analyses, we show that these plasmids belong to a group of 53 P-type T4SS-encoding plasmids in 20 established Acinetobacter species, the majority of clinical relevance, including diverse A. baumannii sequence types and one strain of Providencia rettgeri. The strains were globally distributed in 14 countries spanning five continents, and the conjugative operon's T4SS proteins were highly conserved in most plasmids. A high proportion of plasmids harbored resistance genes, with 17 different genes spanning seven drug classes. Collectively, this demonstrates that P-type T4SS-encoding plasmids are more widespread among the Acinetobacter genus than previously anticipated, including strains of both clinical and environmental importance. This research provides insight into the spread of resistance genes among Acinetobacter and highlights a group of plasmids of importance for future surveillance.

Analytical Challenges in Novel Pentavalent Meningococcal Conjugate Vaccine (A, C, Y, W, X).

Multivalent meningococcal conjugate vaccines are a significant focus for the scientific community in light of the WHO's mission to defeat meningitidis by 2030. Well-known meningococcal vaccines such as MenAfriVac, Nimenrix, Menveo, and MenQuadfi are licensed in various parts of the world and have been successful. Recently, the World Health Organization (WHO) qualified MenFive (meningococcal A, C, Y, W, and X) conjugate vaccine, further enhancing the battery of vaccines against meningitis. The antigenic nature of the current and new serogroups, the selection of carrier proteins, and the optimal formulation of these biomolecules are pivotal parameters for determining whether a biological preparation qualifies as a vaccine candidate. Creating appropriate quality control analytical tools for a complex biological formulation is challenging. A scoping review aims to identify the main challenges and gaps in analyzing multivalent vaccines, especially in the case of novel serogroups, such as X, as the limited literature addresses these analytical challenges. In summary, the similarities in polysaccharide backbones between meningococcal serogroups (C, Y, W sharing a sialic acid backbone and A, X sharing a phosphorous backbone) along with various conjugation chemistries (such as CNBr activation, reductive amination, CDAP, CPIP, thioether bond formation, N-hydroxy succinimide activation, and carbodiimide-mediated coupling) resulting into a wide variety of polysaccharide -protein conjugates. The challenge in analyzing carrier proteins used in conjugation (such as diphtheria toxoid, tetanus toxoid, CRM diphtheria protein, and recombinant CRM) is assessing their purity (whether they are monomeric or polymeric in nature as well as their polydispersity). Additional analytical challenges include the impact of excipients, potential interference from serogroups, selection and establishment of standards, age-dependent behavior of biomolecules indicated by molecular size distributions, and process-driven variations. This article explains the analytical insights gained (polysaccharide content, free saccharide, free proteins, MSD) during the development of the MenFive vaccine and highlights the crucial gaps and challenges in testing.

Comparison of Conjugates Obtained Using DMSO and DMF as Solvents in the Production of Polyclonal Antibodies and ELISA Development: A Case Study on Bisphenol A.

When developing immunochemical test systems, it is necessary to obtain specific antibodies. Their quality depends, among other things, on the immunogen used. When preparing hapten-protein conjugates to obtain antibodies for low-molecular-weight compounds, the key factors are the structure of the hapten itself, the presence of a spacer, the size of the carrier protein and the degree of its modification by hapten molecules. This work shows that one additional factor-the conditions for obtaining the hapten-protein conjugate-is overlooked. In this work, we have synthesized conjugates of bisphenol A derivative 4,4-bis(hydroxyphenyl)valeric acid (BVA), the protein carrier soybean trypsin inhibitor (STI), and bovine serum albumin (BSA) in reaction media combining water with two organic solvents: dimethylformamide (DMF) or dimethyl sulfoxide (DMSO). Namely, BSADMF-BVA, STIDMF-BVA, BSADMSO-BVA and STIDMSO-BVA conjugates were obtained. Rabbit polyclonal antibodies against the BSADMF-BVA conjugate demonstrated basically different interactions in the developed ELISA systems using either STIDMF-BVA or STIDMSO-BVA conjugates. The use of the STIDMF-BVA conjugate demonstrated the absence of competition in combination with antisera obtained from BSADMF-BVA in an ELISA. A competitive interaction was observed only with the use of the STIDMSO-BVA conjugate. Under the selected conditions, the detection limit of bisphenol A was 8.3 ng/mL, and the working range of determined concentrations was 18.5-290.3 ng/mL. The obtained data demonstrate the possibility of achieving sensitive immunoassays by simply varying the reaction media for the hapten-protein conjugation, which could provide an additional tool in the development of immunoassays for other low-molecular-weight compounds.

Controlled enzyme cargo loading in engineered bacterial microcompartment shells.

Bacterial microcompartments (BMCs) are nanometer-scale organelles with a protein-based shell that serve to co-localize and encapsulate metabolic enzymes. They may provide a range of benefits to improve pathway catalysis, including substrate channeling and selective permeability. Several groups are working toward using BMC shells as a platform for enhancing engineered metabolic pathways. The microcompartment shell of Haliangium ochraceum (HO) has emerged as a versatile and modular shell system that can be expressed and assembled outside its native host and with non-native cargo. Further, the HO shell has been modified to use the engineered protein conjugation system SpyCatcher-SpyTag for non-native cargo loading. Here, we used a model enzyme, triose phosphate isomerase (Tpi), to study non-native cargo loading into four HO shell variants and begin to understand maximal shell loading levels. We also measured activity of Tpi encapsulated in the HO shell variants and found that activity was determined by the amount of cargo loaded and was not strongly impacted by the predicted permeability of the shell variant to large molecules. All shell variants tested could be used to generate active, Tpi-loaded versions, but the simplest variants assembled most robustly. We propose that the simple variant is the most promising for continued development as a metabolic engineering platform.