Preserved structure and function of human serum albumin self-folded in the oxidative cytoplasm of Escherichia coli.

Human serum albumin (HSA), a polypeptide featuring 17 disulfide bonds, acts as a crucial transport protein in human blood plasma. Its extended circulation half-life, mediated by FcRn (neonatal Fc receptor)-facilitated recycling, positions HSA as an excellent carrier for long-acting drug delivery. However, the conventional method of obtaining HSA from human blood faces limitations due to availability and potential contamination risks, such as blood-borne diseases. This study introduced SHuffle, an oxidative Escherichia coli (E. coli) expression system, for the production of recombinant HSA (rHSA) that spontaneously self-folds into its native conformation. This system ensures precise disulfide bond formation and correct folding of cysteine-rich rHSA, eliminating the need for chaperone co-expression or domain fusion of a folding enhancer. The purified rHSA underwent thorough physicochemical characterization, including mass spectrometry, circular dichroism spectroscopy, intrinsic fluorescence spectroscopy, esterase-like activity assay, and size exclusion chromatography, to assess critical quality attributes. Importantly, rHSA maintained native binding affinity to FcRn and the albumin-binding domain. Collectively, our analyses demonstrated a high comparability between rHSA and plasma-derived HSA. The expression of rHSA in E. coli with an oxidizing cytosol provides a secure and cost-effective approach, enhancing the potential of rHSA for diverse medical applications.

Covalent immobilization of human serum albumin on cellulose acetate membrane for scavenging amyloid beta - A stepping extracorporeal strategy for ameliorating Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by interrupted neurocognitive functions and impaired mental development presumably caused by the accumulation of amyloid beta (Aß) in the form of plaques. Targeting Aß has been considered a promising approach for treating AD. In the current study, human serum albumin (HSA), a natural Aß binder, is covalently immobilized onto the surface of a cellulose acetate (CA) membrane to devise an extracorporeal Aß sequester. The immobilization of HSA at 3.06 ± 0.22 µg/mm2 of the CA membrane was found to be active functionally, as evidenced by the esterase-like activity converting p-nitrophenyl acetate into p-nitrophenol. The green fluorescent protein-Aß (GFP-Aß) fusion protein, recombinantly produced as a model ligand, exhibited characteristics of native Aß. These features include the propensity to form aggregates or fibrils and an affinity for HSA with a dissociation constant (KD) of 0.91 µM. The HSA on the CA membrane showed concentration-dependent sequestration of GFP-Aß in the 1-10-µM range. Moreover, it had a greater binding capacity than HSA immobilized on a commercial amine-binding plate. Results suggest that the covalent immobilization of HSA on the CA surface can be used as a potential platform for sequestering Aß to alleviate AD.

Recent advancements in nanotechnology based drug delivery for the management of cardiovascular disease.

Cardiovascular diseases (CVDs) constitute a predominant cause of both global mortality and morbidity. To address the challenges in the early diagnosis and management of CVDs, there is growing interest in the field of nanotechnology and nanomaterials to develop innovative diagnostic and therapeutic approaches. This review focuses on the recent advancements in nanotechnology-based diagnostic techniques, including cardiac immunoassays (CIA), cardiac circulating biomarkers, cardiac exosomal biomarkers, and molecular Imaging (MOI). Moreover, the article delves into the exciting developments in nanoparticles (NPs), biomimetic NPs, nanofibers, nanogels, and nanopatchs for cardiovascular applications. And discuss how these nanoscale technologies can improve the precision, sensitivity, and speed of CVD diagnosis and management. While highlighting their vast potential, we also address the limitations and challenges that must be overcome to harness these innovations successfully. Furthermore, this review focuses on the emerging opportunities for personalized and effective cardiovascular care through the integration of nanotechnology, ultimately aiming to reduce the global burden of CVDs.

Streamlined construction of robust heteroprotein complexes by self-induced in-cell disulfide pairing.

Biomolecules and their functional subdomains are essential building blocks in the creation of multifunctional nanocomplexes. Methyl-binding domain protein 2 (MBD2) and p66α stand out as small α-helical motifs with an ability to self-assemble into a heterodimeric coiled-coil, making them promising building units. Yet, their practical use is hindered by rapid dissociation upon dilution. In this study, novel fusion tags, MBD2 and p66α variants, were developed to covalently link during co-expression in E. coli SHuffle. Through strategic placement of cysteine at each α-helix's terminus, intracellular crosslinking occurred with high specificity and yield, facilitated by preserved α-helical interactions. This instant disulfide bonding in the oxidative cytoplasm of E. coli SHuffle efficiently overcame the need for inefficient in vitro oxidation and protein extraction prone to creating non-specific adducts and suboptimal bioprocesses. In contrast to their wild-type counterparts, the GFP-mCherry protein complex cross-linked by the fusion tags maintained the heterodimeric state even under extensive dilution. The fusion tags, when combined with the E. coli SHuffle system, allowed for the streamlined co-expression of a stable protein complex through self-induced intracellular cysteine coupling. The approach demonstrated herein holds great promise for producing multifunctional and robust heteroprotein complexes.

Human serum albumin binders: A piggyback ride for long-acting therapeutics.

Human serum albumin (HSA) is the most abundant protein in the blood and has desirable properties as a drug carrier. One of the most promising ways to exploit HSA as a carrier is to append an albumin-binding moiety (ABM) to a drug for in situ HSA binding upon administration. Nature- and library-derived ABMs vary in size, affinity, and epitope, differentially improving the pharmacokinetics of an appended drug. In this review, we evaluate the current state of knowledge regarding various aspects of ABMs and the unique advantages of ABM-mediated drug delivery. Furthermore, we discuss how ABMs can be specifically modulated to maximize potential benefits in clinical development.

Development and evaluation of bioinspired pH-responsive sericin-chitosan-based hydrogel for controlled colonic delivery of PETase: Harnessing PETase-triggered degradation of microplastics.

The gravity of threats posed by microplastic pollution to the environment cannot be overestimated. Being ubiquitous in the living environment, microplastics reach humans through the food chain causing various hazardous effects. Microplastics can be effectively degraded by PETase enzymes. The current study reports, for the first time, a hydrogel-encapsulated, bioinspired colonic delivery of PETase. A free radical polymerization-assisted hydrogel system was synthesized from sericin, chitosan, and acrylic acid using N,N'-methylenebisacrylamide as a crosslinker and ammonium persulfate as an initiator. The hydrogel was characterized with FTIR, PXRD, SEM, and thermal analysis to confirm the development of a stabilized hydrogel system. The hydrogel showed 61 % encapsulation efficiency, maximum swelling, and cumulative PETase release (96 %) at pH 7.4. The mechanism of PETase release exhibited the Higuchi pattern of release with an anomalous transport mechanism. SDS-PAGE analysis confirmed the preservation of the post-release structural integrity of PETase. The released PETase exhibited concentration- and time-dependent degradation of polyethylene terephthalate in vitro. The developed hydrogel system exhibited the intended features of a stimulus-sensitive carrier system that can be efficiently used for the colonic delivery of PETase.

Unveiling the biological interface of protein complexes by mass spectrometry-coupled methods.

Most biomolecules become functional and bioactive by forming protein complexes through interaction with ligands that are diverse in size, shape, and physicochemical properties. In the complex biological milieu, the interaction is ligand-specific, driven by molecular sensing, and involves the recognition of a binding interface localized within a protein structure. Mapping interfaces of protein complexes is a highly sought area of research as it delivers fundamental insights into proteomes and pathology and hence strategies for therapeutics. While X-ray crystallography and electron microscopy remain the gold standard for structural elucidation of protein complexes, their artificial and static analytic nature often produces a non-native interface that otherwise might be negligible or non-existent in a biological environment. Recently, the mass spectrometry-coupled approaches, chemical crosslinking (CLMS) and hydrogen-deuterium exchange (HDMS) have become valuable analytic complements to the traditional techniques. These methods explicitly identify hot residues and motifs embedded in binding interfaces, especially when the interaction is predominantly dynamic, transient, and/or caused by an intrinsically disordered domain. Here, we review the principal role of CLMS and HDMS in protein structural biology with a particular emphasis on the contribution of recent examples to exploring biological interfaces. Additionally, we describe recent studies that utilized these methods to expand our understanding of protein complex formation and the related biological processes, to increase the probability of structure-based drug design.

Plant extract-based synthesis of metallic nanomaterials, their applications, and safety concerns.

Nanotechnology has attracted the attention of researchers from different scientific fields because of the escalated properties of nanomaterials (NMs) compared with the properties of macromolecules. NMs can be prepared through different approaches involving physical and chemical methods. The development of NMs through plant-based green chemistry approaches is more advantageous than other methods from the perspectives of environmental safety, animal, and human health. The biomolecules and metabolites of plants act as reducing and capping agents for the synthesis of metallic green NMs. Plant-based synthesis is a preferred approach as it is not only cost-effective, easy, safe, clean, and eco-friendly but also provides pure NMs in high yield. Since NMs have antimicrobial and antioxidant potential, green NMs synthesized from plants can be used for a variety of biomedical and environmental remediation applications. Past studies have focused mainly on the overall biogenic synthesis of individual or combinations of metallic NMs and their oxides from different biological sources, including microorganisms and biomolecules. Moreover, from the viewpoint of biomedical applications, the literature is mainly focusing on synthetic NMs. Herein, we discuss the extraction of green molecules and recent developments in the synthesis of different plant-based metallic NMs, including silver, gold, platinum, palladium, copper, zinc, iron, and carbon. Apart from the biomedical applications of metallic NMs, including antimicrobial, anticancer, diagnostic, drug delivery, tissue engineering, and regenerative medicine applications, their environmental remediation potential is also discussed. Furthermore, safety concerns and safety regulations pertaining to green NMs are also discussed.

Bioinspired tunable hydrogels: An update on methods of preparation, classification, and biomedical and therapeutic applications.

Hydrogels exhibit water-insoluble three-dimensional polymeric networks capable of absorbing large amounts of biological fluids. Both natural and synthetic polymers are used for the preparation of hydrogel networks. Such polymeric networks are fabricated through chemical or physical mechanisms of crosslinking. Chemical crosslinking is accomplished mainly through covalent bonding, while physical crosslinking involves self-healing secondary forces like H-bonding, host-guest interactions, and antigen-antibody interactions. The building blocks of the hydrogels play an important role in determining the mechanical, biological, and physicochemical properties. Hydrogels are used in a variety of biomedical applications like diagnostics (biodetection and bioimaging), delivery of therapeutics (drugs, immunotherapeutics, and vaccines), wound dressing and skin materials, cardiac complications, contact lenses, tissue engineering, and cell culture because of the inherent characteristics like enhanced water uptake and structural similarity with the extracellular matrix (ECM). This review highlights the recent trends and advances in the roles of hydrogels in biomedical and therapeutic applications. We also discuss the classification and methods of hydrogels preparation. A brief outlook on the future directions of hydrogels is also presented.

Nanotechnology as a promising platform for rheumatoid arthritis management: Diagnosis, treatment, and treatment monitoring.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that develops in about 5 per 1000 people. Over the past years, substantial progresses in knowledge of the disease's pathophysiology, effective diagnosis methods, early detection, and efficient treatment strategies have been made. Notably, nanotechnology has emerged as a game-changer in the efficacious management of many diseases, especially for RA. Joint replacement, photothermal therapy (PTT), photodynamic therapy (PDT), RA diagnosis, and treatment monitoring are nano-based avenues in RA management. Here, we present a brief overview of the pathogenesis of RA, risk factors, conventional diagnostic methods and treatment approaches, and then discuss the role of nanomedicine in RA diagnosis, treatment, and treatment monitoring with an emphasis on functional characteristics distinctive from other RA therapeutics.

Site-specific proximity ligation provides molecular insights into biologically relevant interfaces of protein-protein interaction.

Dynamic protein-protein interactions (PPIs) are fundamental to spatiotemporal control of protein functions in biological systems. Dissecting binding interfaces in aqueous solution (i.e., biological interfaces) is of great importance for identifying molecular determinants that contribute to the affinity and specificity of PPIs. Herein, we describe a biochemical method, termed site-specific proximity ligation (SPL), that enables the identification and reconstruction of native binding interfaces distinct from those present in crystal structures and models from computational prediction. SPL involves the strategic incorporation of an aryl azide-containing unnatural amino acid (AZF) into residues of interest in a particular protein that forms a multiprotein complex. Depending on the interfacial role of a targeted residue, a photo-inducible highly reactive incorporated AZF moiety may react with neighboring functional groups to covalently capture an otherwise non-covalent or weak interaction with a specific partner protein, thereby revealing the landscape of biological interfaces. Using a heterotrimeric nuclear pore protein as a model, we show that the biological interfaces of the complex mapped by SPL provide new insight into dynamic molecular recognition that is missed by, or even in conflict with, static models.

Fine-tuning bispecific therapeutics.

Bispecific therapeutics target two distinct antigens simultaneously and provide novel functionalities that are not attainable with single monospecific molecules or combinations of them. The unique potential of bispecific therapeutics is driving extensive efforts to discover synergistic dual targets, design molecular formats to integrate bispecific elements, and accelerate successful clinical translation. In particular, the past decade has witnessed a boom in the design and development of bispecific antibody formats with more than 100 collections to date. Despite the remarkable progress that has been made to expand the number of formats, qualitative fine-tuning of bispecific formats is needed to achieve optimal dual-target engagement based on understanding of the spatiotemporal interdependence of the two physically linked binding specificities and the complex target biology associated with bispecific approaches. This review provides insights into the design parameters - including affinity, valency, and geometry - that need to be considered at an early stage of development in order to take the best advantage of bispecific therapeutics.

Site-specific bioconjugation and self-assembly technologies for multi-functional biologics: on the road to the clinic.

The expanding portfolio of biotherapeutics both in the research and development (R&D) and market sectors is shaping new opportunities towards multifunctional biologics (MFBs). The combination of new or pre-existing therapeutic agents into a single multifunctional format makes it possible to develop new pharmacological actions to significantly improve their efficacy and safety. In this review, I focus on novel platform technologies that are being exploited in the biotech industry to produce MFBs with potential therapeutic benefits that include half-life extension, targeted delivery, T cell engagement, and improved vaccination. In this regard, technologies of key importance are site-specific bioconjugation and self-assembly, which allow homogeneous, defined, and scalable process developments for several MFBs that are advancing towards clinical applications.

Conjugation of Cell-Penetrating Peptides to Antimicrobial Peptides Enhances Antibacterial Activity.

Antimicrobial peptides (AMPs), essential elements in host innate immune defenses against numerous pathogens, have received considerable attention as potential alternatives to conventional antibiotics. Most AMPs exert broad-spectrum antimicrobial activity through depolarization and permeabilization of the bacterial cytoplasmic membrane. Here, we introduce a new approach for enhancing the antibiotic activity of AMPs by conjugation of a cationic cell-penetrating peptide (CPP). Interestingly, CPP-conjugated AMPs elicited only a 2- to 4-fold increase in antimicrobial activity against Gram-positive bacteria, but showed a 4- to 16-fold increase in antimicrobial activity against Gram-negative bacteria. Although CPP-AMP conjugates did not significantly increase membrane permeability, they efficiently translocated across a lipid bilayer. Indeed, confocal microscopy showed that, while AMPs were localized mainly in the membrane of Escherichia coli, the conjugates readily penetrated bacterial cells. In addition, the conjugates exhibited a higher affinity for DNA than unconjugated AMPs. Collectively, we demonstrate that CPP-AMP conjugates possess multiple functional properties, including membrane permeabilization, membrane translocation, and DNA binding, which are involved in their enhanced antibacterial activity against Gram-negative bacteria. We propose that conjugation of CPPs to AMPs may present an effective approach for the development of novel antimicrobials against Gram-negative bacteria.

Enhanced expression of soluble antibody fragments by low-temperature and overdosing with a nitrogen source.

Escherichia coli has been a primary host for the prokaryotic production of antibody fragments (Fabs) and has contributed to several successes in the pharmaceutical industry. Nevertheless, the requirement of disulfide bonds often results in low-yield fermentation and a lack of cost-effectiveness. Despite the improved production of functional Fabs by fermentation below 30 °C, the limited cellular growth needs further work. To address these issues, we investigated the effect of nitrogen supply on the cellular growth and the Fab productivity. We used the anti-human VEGF-A Fab as a model that exhibited poor expression at 37 °C regardless of the amount of nitrogen supplied during fermentation. In stark contrast, the expression yield of soluble Fab with a gross nitrogen supply of 6.91 g/L of broth throughout the fermentation at 25 °C was 332 mg/L. Furthermore, and increased nitrogen supply of 10.9 g/L significantly improved the yield of active form by 59.7% and the cellular growth rate by 39.3%. These results indicate that overdosing of a nitrogen source at low temperature is critical to Fab productivity in E. coli.

Generation of therapeutic protein variants with the human serum albumin binding capacity via site-specific fatty acid conjugation.

Extension of the serum half-life is an important issue in developing new therapeutic proteins and expanding applications of existing therapeutic proteins. Conjugation of fatty acid, a natural human serum albumin ligand, to a therapeutic protein/peptide was developed as a technique to extend the serum half-life in vivo by taking advantages of unusually long serum half-life of human serum albumin (HSA). However, for broad applications of fatty acid-conjugation, several issues should be addressed, including a poor solubility of fatty acid and a substantial loss in the therapeutic activity. Therefore, herein we systematically investigate the conditions and components in conjugation of fatty acid to a therapeutic protein resulting in the HSA binding capacity without compromising therapeutic activities. By examining the crystal structure and performing dye conjugation assay, two sites (W160 and D112) of urate oxidase (Uox), a model therapeutic protein, were selected as sites for fatty acid-conjugation. Combination of site-specific incorporation of a clickable p-azido-L-phenylalanine to Uox and strain-promoted azide-alkyne cycloaddition allowed the conjugation of fatty acid (palmitic acid analog) to Uox with the HSA binding capacity and retained enzyme activity. Deoxycholic acid, a strong detergent, greatly enhanced the conjugation yield likely due to the enhanced solubility of palmitic acid analog.

Self-assembled protein nanocarrier for intracellular delivery of antibody.

Despite the great potential of antibodies as intracellular therapeutics, there is a significant, unmet challenge in delivering sufficient amounts of folded antibodies inside cells. We describe an all-protein self-assembled nanocarrier capable of delivering functional antibodies to the cytosol. By combining an α-helical peptide that self-assembles into a hexameric coiled-coil bundle and an Fc-binding Protein A fragment, we generated the Hex nanocarrier that is efficiently internalized by cells without cytotoxicity. Localization of multiple Fc-binding domains on the hexameric core allowed the Hex nanocarrier to tightly bind antibody with sub-nanomolar affinity regardless of pH and the antibody's originating species. The size of the Hex nanocarrier ranges from 25 to 35nm depending on the antibody loading ratio. We demonstrated the capacity of the Hex nanocarrier to deliver functional antibodies to the cytosol by employing anti-ß-tubulin or anti-nuclear pore complex antibody as cargo. The design of the Hex nanocarrier is modular, which enables functionalization beyond Fc-binding. We exploited this feature to improve the cytosolic delivery efficiency of the Hex nanocarrier by addition of an endosomolytic motif to the core. The modified Hex nanocarrier exhibited similar antibody-binding behavior, but delivered more antibodies to their cytosolic targets at a faster rate. This work demonstrates an efficient intracellular antibody delivery platform with significant advantages over existing approaches as it does not require modification of the antibody, is biodegradable, and has an antibody to carrier mass ratio of 13, which is greater than other reported antibody carriers.

Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex.

Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other.

Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein.

Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. However, non-specific multi-site fluorescent labeling often results in a loss of native structure and function, and single cysteine labeling is not feasible when native cysteines are required to support a protein's folding or catalytic activity. Here, we develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site-specifically label with a single fluorescent small molecule or protein the myristoyl-switch protein recoverin, which is involved in rhodopsin-mediated signaling in mammalian visual sensory neurons. We demonstrate reversible Ca(2+)-responsive translocation of labeled recoverin to membranes and show that recoverin favors membranes with negative curvature and high lipid fluidity in complex heterogeneous membranes, which confers spatio-temporal control over down-stream signaling events. The site-specific orthogonal labeling technique is promising for structural, dynamical, and functional studies of many lipid-anchored membrane protein switches.

Site-Specific Albumination as an Alternative to PEGylation for the Enhanced Serum Half-Life in Vivo.

Polyethylene glycol (PEG) has been widely used as a serum half-life extender of therapeutic proteins. However, due to immune responses and low degradability of PEG, developing serum half-life extender alternatives to PEG is required. Human serum albumin (HSA) has several beneficial features as a serum half-life extender, including a very long serum half-life, good degradability, and low immune responses. In order to further evaluate the efficacy of HSA, we compared the extent of serum half-life extension of a target protein, superfolder green fluorescent protein (sfGFP), upon HSA conjugation with PEG conjugation side-by-side. Combination of site-specific incorporation of p-azido-l-phenylalanine into sfGFP and copper-free click chemistry achieved the site-specific conjugation of a single HSA, 20 kDa PEG, or 30 kDa PEG to sfGFP. These sfGFP conjugates exhibited the fluorescence comparable to or even greater than that of wild-type sfGFP (sfGFP-WT). In mice, HSA-conjugation to sfGFP extended the serum half-life 9.0 times compared to that of unmodified sfGFP, which is comparable to those of PEG-conjugated sfGFPs (7.3 times for 20 kDa PEG and 9.5 times for 30 kDa PEG). These results clearly demonstrated that HSA was as effective as PEG in extending the serum half-life of a target protein. Therefore, with the additional favorable features, HSA is a good serum half-life extender of a (therapeutic) protein as an alternative to PEG.