Affinity purification/immobilization of poly histidine-tagged proteins by nickel-functionalized porous chitosan membranes.

Although immobilized metal ion affinity chromatography (IMAC) is one of the most effective methods for purifying his-tagged proteins, it has limitations such as expensive commercial resins and non-specific binding of unwanted proteins to the nickel immobilized on the resin. In this study, biocompatible chitosan and porous chitosan membranes as alternative resins were synthesized for protein immobilization and purification, but finally porous chitosan membrane was selected due to its higher porosity and consequently higher nickel adsorption. Once the membrane was functionalized with nickel ions and its metal adsorption confirmed by EDS and ICP methods, it was used to immobilize and purify recombinant ß-NGF as a protein model with his-tag tail in batch-fashion. Protein binding and purification were also approved by FTIR and UV-Vis spectroscopy and SDS-PAGE technique. Our results indicated that the protein of interest could bind to the nickel-functionalized porous chitosan membrane with high efficiency at pH=7. Furthermore, for protein purification, the pH value of 6 and an imidazole concentration of 750 mM were suggested for the final elution buffer. In conclusion, nickel-functionalized porous chitosan membrane could be a suitable alternative to IMAC for low cost and specific protein immobilization and purification.

Highly efficient GPCR immobilization with enhanced fouling resistance, salt tolerance, and chromatographic performance.

The feasibility of immobilized protein-based biodetection relies critically on the activity of the immobilized proteins as well as the biocompatibility of the protein surface. Although many protein immobilization strategies have been developed with satisfied detection readout signals. Non-specific interactions caused by the protein-coating surface are still of great concern since they often interfere with or affect the reliability of detection. Herein, we developed a highly efficient G protein-coupled receptor (GPCR) immobilization method by the combination of polyethylene glycol (PEG) with a self-labeling enzyme-catalyzed reaction. The immobilization relies on the covalent interaction between the fusion tag of a target GPCR (kinase domain of epidermal growth factor receptor, EGFR) and its covalent inhibitor ibrutinib, which is modified on PEGylated silica gels. Two types of GPCRs, N-methyl-D-aspartate 2 A receptor (NMDAR2A) and endothelin A receptor (ETAR), were used as examples to realize protein immobilization. The GPCR modified gels and the affinity columns packed with them have been extensively characterized, in terms of non-specific adsorptions, retention factor (k'), half peak width (W1/2), tailing factor (Tf), theoretical plates (N), and association and dissociation constants of the ligands with the receptors. The immobilized GPCRs with reduced non-specific interactions and enhanced fouling resistance, salt tolerance, and chromatographic performance were clearly observed. We believe it is the first work to introduce PEGylation in GPCR immobilization and provide comprehensive proof-of-concept studies to illustrate the improved antifouling property, salt tolerance, and chromatographic performance. This method could be generally applicable in other immobilized protein-based technology for reliable biodetection.

Actinomycin-X2-Immobilized Silk Fibroin Film with Enhanced Antimicrobial and Wound Healing Activities.

Actinomycin is a family of chromogenic lactone peptides that differ in their peptide portions of the molecule. An antimicrobial peptide, actinomycin X2 (Ac.X2), was produced through the fermentation of a Streptomyces cyaneofuscatus strain. Immobilization of Ac.X2 onto a prepared silk fibroin (SF) film was done through a carbodiimide reaction. The physical properties of immobilized Ac.X2 (antimicrobial films, AMFs) were analyzed by ATR-FTIR, SEM, AFM, and WCA. The findings from an in vitro study showed that AMFs had a more broad-spectrum antibacterial activity against both S. aureus and E. coli compared with free Ac.X2, which showed no apparent strong effect against E. coli. These AMFs showed a suitable degradation rate, good hemocompatibility, and reduced cytotoxicity in the biocompatibility assay. The results of in vivo bacterially infected wound healing experiments indicated that wound inflammation was prevented by AMFs, which promoted wound repair and improved the wound microenvironment. This study revealed that Ac.X2 transformation is a potential candidate for skin wound healing.

Single-molecule conformational dynamics of a transcription factor reveals a continuum of binding modes controlling association and dissociation.

Binding and unbinding of transcription factors to DNA are kinetically controlled to regulate the transcriptional outcome. Control of the release of the transcription factor NF-κB from DNA is achieved through accelerated dissociation by the inhibitor protein IκBα. Using single-molecule FRET, we observed a continuum of conformations of NF-κB in free and DNA-bound states interconverting on the subseconds to minutes timescale, comparable to in vivo binding on the seconds timescale, suggesting that structural dynamics directly control binding kinetics. Much of the DNA-bound NF-κB is partially bound, allowing IκBα invasion to facilitate DNA dissociation. IκBα induces a locked conformation where the DNA-binding domains of NF-κB are too far apart to bind DNA, whereas a loss-of-function IκBα mutant retains the NF-κB conformational ensemble. Overall, our results suggest a novel mechanism with a continuum of binding modes for controlling association and dissociation of transcription factors.

Oral Administration of Resveratrol-Selenium-Peptide Nanocomposites Alleviates Alzheimer's Disease-like Pathogenesis by Inhibiting Aß Aggregation and Regulating Gut Microbiota.

Alzheimer's disease (AD) is a neurodegenerative disease associated with amyloid-ß (Aß) deposition, leading to neurotoxicity (oxidative stress and neuroinflammation) and gut microbiota imbalance. Resveratrol (Res) has neuroprotective properties, but its bioavailability in vivo is very low. Herein, we developed a small Res-selenium-peptide nanocomposite to enable the application of Res for eliminating Aß aggregate-induced neurotoxicity and mitigating gut microbiota disorder in aluminum chloride (AlCl3) and d-galactose(d-gal)-induced AD model mice. Res functional selenium nanoparticles (Res@SeNPs) (8 ± 0.34 nm) were prepared first, after which the surface of Res@SeNPs was decorated with a blood-brain barrier transport peptide (TGN peptide) to generate Res-selenium-peptide nanocomposites (TGN-Res@SeNPs) (14 ± 0.12 nm). Oral administration of TGN-Res@SeNPs improves cognitive disorder through (1) interacting with Aß and decreasing Aß aggregation, effectively inhibiting Aß deposition in the hippocampus; (2) decreasing Aß-induced reactive oxygen species (ROS) and increasing activity of antioxidation enzymes in PC12 cells and in vivo; (3) down-regulating Aß-induced neuroinflammation via the nuclear factor kappa B/mitogen-activated protein kinase/Akt signal pathway in BV-2 cells and in vivo; and (4) alleviating gut microbiota disorder, particularly with respect to oxidative stress and inflammatory-related bacteria such as Alistipes, Helicobacter, Rikenella, Desulfovibrio, and Faecalibaculum. Thus, we anticipate that Res-selenium-peptide nanocomposites will offer a new potential strategy for the treatment of AD.

Magnetic Nanoplatforms for Covalent Protein Immobilization Based on Spy Chemistry.

Immobilization of proteins on magnetic nanoparticles (MNPs) is an effective approach to improve protein stability and facilitate separation of immobilized proteins for repeated use. Herein, we exploited the efficient SpyTag-SpyCatcher chemistry for conjugation of functional proteins onto MNPs and established a robust magnetic-responsive nanoparticle platform for protein immobilization. To maximize the loading capacity and achieve outstanding water dispersity, the SpyTag peptide was incorporated into the surface-charged polymers of MNPs, which provided abundant active sites for Spy chemistry while maintaining excellent colloidal stability in buffer solution. Conjugation between enhanced green fluorescence protein (EGFP)-SpyCatcher-fused proteins and SpyTag-functionalized MNPs was efficient at ambient conditions without adding enzymes or chemical cross-linkers. Benefiting from the excellent water dispersity and interface compatibility, the surface Spy reaction has fast kinetics, which is comparable to that of the solution Spy reaction. No activity loss was observed on EGFP after conjugation due to the site-selective nature of Spy chemistry. The immobilization process of EGFP on MNPs was highly specific and robust, which was not affected by the presence of other proteins and detergents, such as bovine serum albumin and Tween 20. The MNP platform was demonstrated to be protective to the conjugated EGFP and significantly improved the shelf life of immobilized proteins. In addition, experiments confirmed the retained magnetophoresis of the MNP after protein loading, demonstrating fast MNP recovery under an external magnetic field. This MNP is expected to provide a versatile and modular platform to achieve effective and specific immobilization of other functional proteins, enabling easy reuse and storage.

Atomically Resolved Characterization of Optically Driven Ligand Reconfiguration on Nanoparticle Catalyst Surfaces.

Dynamic ligand layers on nanoparticle surfaces could prove to be critically important to enhance the functionality of individual materials. Such capabilities could complement the properties of the inorganic component to provide multifunctionality or the ability to be remotely actuated. Peptide-based ligands have demonstrated the ability to be remotely responsive to structural changes when adsorbed to nanoparticle surfaces via incorporation of photoswitches into their molecular structure. In this contribution, direct spectroscopic evidence of the remote actuation of a photoswitchable peptide adsorbed onto Au nanoparticles is demonstrated using X-ray absorption fine structure spectroscopic methods. From this analysis, Au-X (X = C or N) coordination numbers confirm the changes before and after photoswitching in the surface ligand conformation, which was correlated directly to variations in the catalytic application of the materials for nitrophenol reduction processes. In addition, the catalytic application of the materials was demonstrated to be significantly sensitive to the structure of the nitrophenol substrate used in the reaction, suggesting that changes in the reactivity are likely based upon the peptide conformation and substrate structure. Such results confirm that surface ligands can be remotely reconfigured on nanoparticle surfaces, providing pathways to apply such capabilities to a variety of applications beyond catalysis ranging from drug delivery to sensing.

Photolithographic Patterning of FluorAcryl for Biphilic Microwell-Based Digital Bioassays and Selection of Bacteria.

FluorAcryl 3298 (FA) is a UV-curable fluoroacrylate polymer commonly employed as a chemically resistant, hydrophobic, and oleophobic coating. Here, FA was used in a cleanroom-based microstructuring process to fabricate hydrophilic-in-hydrophobic (HiH) micropatterned surfaces containing femtoliter-sized well arrays. A short protocol involving direct UV photopatterning, an etching step, and final recovery of the hydrophobic properties of the polymer produced patterned substrates with micrometer resolution. Specifically, HiH microwell arrays were obtained with a well diameter of 10 µm and various well depths ranging from 300 nm to 1 µm with high reproducibility. The 300 nm deep microdroplet array (MDA) substrates were used for digital immunoassays, which presented a limit of detection in the attomolar range. This demonstrated the chemical functionality of the hydrophilic and hydrophobic surfaces. Furthermore, the 1 µm deep wells could efficiently capture particles such as bacteria, whereas the 300 nm deep substrates or other types of flat HiH molecular monolayers could not. Capturing a mixture of bacteria expressing red- and green-fluorescent proteins, respectively, served as a model for screening and selection of specific phenotypes using FA-MDAs. Here, green-fluorescent bacteria were specifically selected by overlaying a solution of gelatin methacryloyl (GelMA) mixed with a photoinitiator and using a high-magnification objective, together with custom pinholes, in a common fluorescence microscope to cross-link the hydrogel around the bacteria of interest. In conclusion, due to the straightforward processing, versatility, and low-price, FA is an advantageous alternative to more commonly used fluorinated materials, such as CYTOP or Teflon-AF, for the fabrication of HiH microwell arrays and other biphilic microstructures.

A Synergistic New Approach Toward Enhanced Antibacterial Efficacy via Antimicrobial Peptide Immobilization on a Nitric Oxide-Releasing Surface.

Despite technological advancement, nosocomial infections are prevalent due to the rise of antibiotic resistance. A combinatorial approach with multimechanistic antibacterial activity is desired for an effective antibacterial medical device surface strategy. In this study, an antimicrobial peptide, nisin, is immobilized onto biomimetic nitric oxide (NO)-releasing medical-grade silicone rubber (SR) via mussel-inspired polydopamine (PDA) as a bonding agent to reduce the risk of infection. Immobilization of nisin on NO-releasing SR (SR-SNAP-Nisin) and the surface characteristics were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy with energy-dispersive X-ray spectroscopy and contact angle measurements. The NO release profile (7 days) and diffusion of SNAP from SR-SNAP-Nisin were quantified using chemiluminescence-based nitric oxide analyzers and UV-vis spectroscopy, respectively. Nisin quantification showed a greater affinity of nisin immobilization toward SNAP-doped SR. Matrix-assisted laser desorption/ionization mass spectrometry analysis on surface nisin leaching for 120 h under physiological conditions demonstrated the stability of nisin immobilization on PDA coatings. SR-SNAP-Nisin shows versatile in vitro anti-infection efficacy against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus in the planktonic and adhered states. Furthermore, the combination of NO and nisin has a superior ability to impair biofilm formation on polymer surfaces. SR-SNAP-Nisin leachates did not elicit cytotoxicity toward mouse fibroblast cells and human umbilical vein endothelial cells, indicating the biocompatibility of the material in vitro. The preventative and therapeutic potential of SR-SNAP-Nisin dictated by two bioactive agents may offer a promising antibacterial surface strategy.

A Simplistic Single-Step Method for Preparing Biomimetic Nanoparticles from Endogenous Biomaterials.

Many works utilize products isolated from nature as capping agents to functionalize gold nanoparticles for targeting and therapeutic applications. Some of the most advanced of these strategies utilize complex multicomponent biomaterials, such as whole cell-membranes, for nanoparticle functionalization strategies for evading or initializing immune response as well as for targeting. Strategies like these, wherein whole cell membrane is utilized for functionalization, take advantage of the complexity of the protein-lipid content and organization, which cells normally use for communication and interaction (instilling these capacities to nanoparticle vectors). Many approaches for achieving this in functionalizing the surface of nanoparticles rely on multistep processes, which necessitate the addition and then removal of synthetic molecules, heating, or pH modifications. These processes can have deleterious modifying effects on the functionalizing biomolecules, resulting in loss of product and time during each purification step, as well as potentially changing the biomolecule functionality toward a nondesirable effect. Here, we describe methods for forming gold nanoparticles at room temperature in a single step, functionalized with proteins, using nicotinamide adenine dinucleotide (NADH). This process enables formation of nanoparticles that can be functionalized by individual proteins (demonstrated with FBS) or whole cells membrane (extracted from B16F10 cells). This work is derivative from observations found in the literature by us and others, that mammalian cells are capable of producing gold nanoparticles from ionic gold without the supplementation of chemical species. The products of this single-step synthesis described herein have been optimized to maintain biomolecule integrity and so that there are no further purification steps required. To characterize the nanoparticles in terms of their shape, size, surface functionality, and biomolecule integrity throughout development, we employed light-based spectroscopy techniques, molecular modeling, electron microscopy, light scattering, and gel electrophoresis techniques. In order to compare the optimized biomolecule-functionalized nanoparticles against current standards (which require synthetic linkers, heating, or pH manipulation), we employed metabolic and live/dead assays as well as light-based microscopy/spectroscopy in vitro. In comparing our synthetic process against others for forming gold nanoparticles functionalized with complex biomolecule components (whole-cell membrane), we found that this process had superior particle internalization. Our strategy has similar outlets for application to these other works, however, because this process is entirely reliant on endogenous biomaterials and has additional potential.

Recent advances in nanomaterials-based electrochemical immunosensors and aptasensors for HER2 assessment in breast cancer.

Human epidermal growth factor receptor 2 (HER2) is one of the key molecular targets in breast cancer pathogenesis. Overexpression and/or amplification of HER2 in approximately 15-20% of breast cancer patients is associated with high mortality and poor prognosis. Accumulating evidence shows that accurate and sensitive detection of HER2 improves the survival outcomes for HER2-positive breast cancer patients from targeted therapies. The current methods of clinical determination of HER2 expression levels are based on slide-based assays that rely on invasively collected primary tumours. Alternatively, ELISA-based detection of the shredded HER2 extracellular domain (HER2-ECD) of has been suggested as a surrogate method for monitoring disease progress and treatment response in breast cancer patients. In the past decade, biosensors have emerged as an alternative modality for the detection of circulating HER2-ECD in human serum samples. In particular, electrochemical biosensors based on nanomaterials and antibodies and aptamers have been increasingly developed as promising tools for rapid, sensitive, and cost-effective detection of HER2-ECD. These biosensors harness the high affinity and specificity of antibodies and aptamers, and unique conductive properties, biocompatibility, large surface area, and chemical stability of nanomaterials for selective and sensitive assessment of the HER2. This review provides an overview of the recent advances in the application of nanomaterials-based immunosensors and aptasensors for detection of circulating HER2-ECD. In particular, various electrochemical techniques, detection approaches, and nanomaterials are discussed. Further, analytical figures of merit of various HER2 immunosensors and aptasensors are compared. Finally, possible challenges and potential opportunities for biosensor-based detection of HER2-ECD are discussed.

Glycan Array Evaluation of Synthetic Epitopes between the Capsular Polysaccharides from Streptococcus pneumoniae 19F and 19A.

Vaccination represents the most effective way to prevent invasive pneumococcal diseases. The glycoconjugate vaccines licensed so far are obtained from capsular polysaccharides (CPSs) of the most virulent serotypes. Protection is largely limited to the specific vaccine serotypes, and the continuous need for broader coverage to control the outbreak of emerging serotypes is pushing the development of new vaccine candidates. Indeed, the development of efficacious vaccine formulation is complicated by the high number of bacterial serotypes with different CPSs. In this context, to simplify vaccine composition, we propose the design of new saccharide fragments containing chemical structures shared by different serotypes as cross-reactive and potentially cross-protective common antigens. In particular, we focused on Streptococcus pneumoniae (Sp) 19A and 19F. The CPS repeating units of Sp 19F and 19A are very similar and share a common structure, the disaccharide ManNAc-ß-(1→4)-Glc (A-B). Herein, we describe the synthesis of a small library of compounds containing different combinations of the common 19F/19A disaccharide. The six new compounds were tested with a glycan array to evaluate their recognition by antibodies in reference group 19 antisera and factor reference antisera (reacting against 19F or 19A). The disaccharide A-B, phosphorylated at the upstream end, emerged as a hit from the glycan array screening because it is strongly recognized by the group 19 antisera and by the 19F and 19A factor antisera, with similar intensity compared with the CPSs used as controls. Our data give a strong indication that the phosphorylated disaccharide A-B can be considered a common epitope among different Sp 19 serotypes.

Bioinspired caries preventive strategy via customizable pellicles of saliva-derived protein/peptide constructs.

Dental caries has been the most widespread chronic disease globally associated with significant health and financial burdens. Caries typically starts in the enamel, which is a unique tissue that cannot be healed or regrown; nonetheless, new preventive approaches have limitations and no effective care has developed yet. Since enamel is a non-renewable tissue, we believe that the intimate overlaying layer, the acquired enamel pellicle (AEP), plays a crucial lifetime protective role and could be employed to control bacterial adhesion and dental plaque succession. Based on our identified AEP whole proteome/peptidome, we investigated the bioinhibitory capacities of the native abundant proteins/peptides adsorbed in pellicle-mimicking conditions. Further, we designed novel hybrid constructs comprising antifouling and antimicrobial functional domains derived from statherin and histatin families, respectively, to attain synergistic preventive effects. Three novel constructs demonstrated significant multifaceted bio-inhibition compared to either the whole saliva and/or its native proteins/peptides via reducing biomass fouling and inducing biofilm dispersion beside triggering bacterial cell death. These data are valuable to bioengineer precision-guided enamel pellicles as an efficient and versatile prevention remedy. In conclusion, integrating complementary acting functional domains of salivary proteins/peptides is a novel translational approach to design multifunctional customizable enamel pellicles for caries prevention.

Simultaneous Stabilization and Functionalization of Gold Nanoparticles via Biomolecule Conjugation: Progress and Perspectives.

Gold nanoparticles (AuNPs) are used in various biological applications because of their small surface area-to-volume ratios, ease of synthesis and modification, low toxicity, and unique optical properties. These properties can vary significantly with changes in AuNP size, shape, composition, and arrangement. Thus, the stabilization of AuNPs is crucial to preserve the properties required for biological applications. In recent years, various polymer-based physical and chemical methods have been extensively used for AuNP stabilization. However, a new stabilization approach using biomolecules has recently attracted considerable attention. Biomolecules such as DNA, RNA, peptides, and proteins are representative of the biomoieties that can functionalize AuNPs. According to several studies, biomolecules can stabilize AuNPs in biological media; in addition, AuNP-conjugated biomolecules can retain certain biological functions. Furthermore, the presence of biomolecules on AuNPs significantly enhances their biocompatibility. This review provides a representative overview of AuNP functionalization using various biomolecules. The strategies and mechanisms of AuNP functionalization using biomolecules are comprehensively discussed in the context of various biological fields.

Photoresponsive Bridged Polysilsesquioxanes for Protein Immobilization/Controlled Release and Micropatterns.

Protein micropatterning on microfabricated surfaces is a promising technology in applications for biochip microarrays, cell attachment, and biosensors. In the present work, a novel photoresponsive polymer based on light-triggered charge shifting bridged polysilsesquioxane (CBPS) is designed and prepared. The organic bridged units containing a photocleavable group of diethylaminocoumarin-4-yl in CBPS could be cleaved rapidly upon irradiation at 410 nm, resulting in the polymer surface switching from a positive charge to a negative charge property. The photoresponsive behavior of CBPS is studied using FTIR, UV-vis, SEM, fluorescence microscopy, and zeta potential analysis. Proteins are easily immobilized on the polymer surface via electrostatic interactions and released after irradiation as required. Combined with photopatterning techniques, accurate protein micropatterns are fabricated by covering a photomask upon irradiation. A gradient protein pattern is also spatially and temporally controlled by regulating irradiation parameters. This smart photoresponsive polymer surface provides a gentle and straightforward strategy to micropattern charged proteins. Moreover, the photoresponsive polymer holds permitting potential in biomedical applications such as conjugating biomolecules, guiding cell arrays, and resisting bacteria.

Application of Engineered Zinc Finger Proteins Immobilized on Paramagnetic Beads for Multiplexed Detection of Pathogenic DNA.

Micro-scale magnetic beads are widely used for isolation of proteins, DNA, and cells, leading to the development of in vitro diagnostics. Efficient isolation of target biomolecules is one of the keys to developing a simple and rapid point-of-care diagnostic. A zinc finger protein (ZFP) is a double-stranded (ds) DNA-binding domain, providing a useful scaffold for direct reading of the sequence information. Here, we utilized two engineered ZFPs (Stx2-268 and SEB-435) to detect the Shiga toxin (stx2) gene and the staphylococcal enterotoxin B (seb) gene present in foodborne pathogens, Escherichia coli O157 and Staphylococcus aureus, respectively. Engineered ZFPs are immobilized on a paramagnetic bead as a detection platform to efficiently isolate the target dsDNA-ZFP bound complex. The small paramagnetic beads provide a high surface area to volume ratio, allowing more ZFPs to be immobilized on the beads, which leads to increased target DNA detection. The fluorescence signal was measured upon ZFP binding to fluorophore-labeled target dsDNA. In this study, our system provided a detection limit of ≤ 60 fmol and demonstrated high specificity with multiplexing capability, suggesting a potential for development into a simple and reliable diagnostic for detecting multiple pathogens without target amplification.

New Tool for Rapid and Accurate Detection of Interleukin-2 and Soluble Interleukin-2 Receptor α in Cancer Diagnosis Using a Bioresponsive Microgel and Multivalent Protein Binding.

Interleukin-2 (IL-2) and its α receptor in soluble form (sIL-2Rα) are considered biomarkers for cancers and immune-related diseases. Enzyme-linked immunosorbent assay is the most common method used to evaluate biomarkers in clinical practice; it is precise but time-consuming and involves complicated procedures. Here, we have developed a rapid yet accurate modality for cancer diagnosis that enables on-site evaluation of cancer markers, that is, IL-2 and sIL-2Rα, without complicated pretreatment of cancer patient-derived blood samples. Surface plasmon resonance and bioresponsive microgels conjugated with IL-2 receptors, that is, IL-2Rß and IL-2Rγ, were utilized to measure IL-2 and sIL-2Rα levels via multivalent protein binding (MPB) between the ligands and their receptors. Our results showed that this novel method enables us to perform cancer diagnosis with a 1000-fold dilution of serum in 10 min. The advantage of MPB-based cancer diagnosis originates from its great selectivity for a target molecule and tolerance to a myriad of nonspecific substances in serum, which allows on-site clinical evaluation. Importantly, our finding implies that MPB-based cancer diagnosis provides a new paradigm not only for improving cancer treatment but also for evaluating a target molecule in unpurified and complex solutions such as blood.

Glycoprotein analysis using lectin microcolumns and capillary electrophoresis: Characterization of alpha1-acid glycoprotein by combined separation methods.

Separations based on combinations of 2.1 mm I.D. high-performance affinity microcolumns and capillary electrophoresis were developed and used to characterize the glycoforms of an intact glycoprotein. Human alpha1-acid glycoprotein (AGP) was used as a model analyte due to its heterogeneous glycosylation resulting from variations in its degree of branching, fucosylation, and number of sialic acids. Three separation formats were examined based on microcolumns that contained the lectins concanavalin A (Con A) or Aleuria aurantia lectin (AAL). These microcolumns were used with one another or in combination with capillary electrophoresis. N-Glycan analysis of the non-retained and retained AGP fractions was carried out by using PNGase F digestion and nanoflow electrospray ionization mass spectrometry. Con A microcolumns were found to selectively enrich AGP that contained bi-antennary N-glycans, while AAL microcolumns retained AGP with fucose-containing N-glycans. Results from these separation methods indicated that fucosylation of the N-linked glycans was more abundant when a high degree of branching was present in AGP. Sialic acid residues were more abundant when higher degrees of branching and more fucose residues were present in AGP. The separation and analysis methods that were developed could be used with relatively small amounts of AGP and can be adapted for use with other intact glycoproteins.

Construction of Metal Hydrate-Based Amorphous Magnetic Nanosheets for Enhanced Protein Enrichment and Immobilization.

Inspired by the hierarchical fabrication technique, many self-assembly procedures have improved the construction of nanomaterials with unique physicochemical characteristics and multiple functions. The generation of multiple complexes is always accompanied by hierarchical structures and intriguing properties that are distinct from their individual segments. An interesting composite is amorphous magnetic Zn-Zr phosphate hydrated nanosheets (Zn-Zr APHNs), generated using templated synthesis and nanoparticle codeposition. The special porous structure of this construct, together with the abundance of metal ions and hydrate present, endows it with many interaction sites for proteins, provides high loading efficiency, and enhances bioactivity. Then, a series of proteins, including enzymes, was immobilized by the Zn-Zr APHNs by multiple interactions, high ionization, and larger surface of the nanosheets. In this study, novel methods for the enrichment of bioactive proteins while retaining the activity of protein payloads are presented. As a verification method, it is indicated that the Zn-Zr APHNs can deliver enzyme proteins (i.e., Cyt-c) to increase the catalytic activity with their biological function and structural integrity, resulting in a highly increased activity to free proteins.

Tailored nanodisc immobilization for one-step purification and reconstitution of cytochrome P450: A tool for membrane proteins' hard cases.

A novel nanodisc-based immobilization method was developed for high-efficient purification and reconstitution of cytochrome P450 in one step. Using membrane scaffold protein containing a histidine tag, charged-nanodiscs were prepared in the form of self-assembly of lipid-protein nanoparticles. Their properties including the particle diameter and its distribution and Zeta potential were controlled well by adjusting molar ratios of phospholipids to membrane scaffold protein. At an optimum lipid-to-membrane scaffold protein molar ratio of 60:1, uniformly regular-shaped and discoidal nanodiscs with an average particle diameter of 10 nm and Zeta potential of -19 mV were obtained. They can be well fractionated by size exclusion chromatography. Charged-nanodiscs were successfully immobilized onto Ni-chelating microspheres via histidine tags with a density of 6.6 mg membrane scaffold protein/mL gel. After being packed in a column, chromatography studies demonstrated that this nanodisc-immobilized chromatographic medium had a specific binding to cytochrome P450 in rat liver microsome. Nanodiscs containing cytochrome P450 can be furthermore eluted from the column with a diameter of about 87.0 nm and height of about 8.0 nm, respectively. The purity of cytochrome P450 after purification increased 25 folds strikingly. This nanodisc-immobilized chromatography method is promising for the one-step purification and reconstitution of membrane protein.