Site-directed Immobilization Research Articles

Directional co-immobilization of artificial multimeric-enzyme complexes as a robust biocatalyst for biosynthesis curcumin glucosides and regeneration of UDP-glucose

Site-directed protein immobilization allows the homogeneous orientation of proteins while maintaining high activity, which is advantageous for various applications. In this study, the use of SpyCatcher/SpyTag technology and magnetic nickel ferrite (NiFe2O4 NPs) nanoparticles were used to prepare a site-directed immobilization of BsUGT2m from Bacillus subtilis and AtSUSm from Arabidopsis thaliana for enhancing curcumin glucoside production with UDP-glucose regeneration from sucrose and UDP. The immobilization of self-assembled multienzyme complex (MESAs) enzymes were characterized for immobilization parameters and stability, including thermal, pH, storage stability, and reusability. The immobilized MESAs exhibited a 2.5-fold reduction in UDP consumption, enhancing catalytic efficiency. Moreover, the immobilized MESAs demonstrated high storage and temperature stability over 21 days at 4 °C and 25 °C, outperforming their free counterparts. Reusability assays showed that the immobilized MESAs retained 78.7 % activity after 10 cycles. Utilizing fed-batch technology, the cumulative titer of curcumin 4’-O-β-D-glucoside reached 6.51 mM (3.57 g/L) and 9.45 mM (5.18 g/L) for free AtSUSm/BsUGT2m and immobilized MESAs, respectively, over 12 h. This study demonstrates the efficiency of magnetic nickel ferrite nanoparticles in co-immobilizing enzymes, enhancing biocatalysts' catalytic efficiency, reusability, and stability.

Site-Directed Immobilization of An Engineered Cytidine Deaminase on a Magnetic Carrier with Bacteriophage-Aided Nanosurface for Multiround Biocatalysis

Site-Directed Immobilization of An Engineered Cytidine Deaminase on a Magnetic Carrier with Bacteriophage-Aided Nanosurface for Multiround Biocatalysis

Direct Affinity Ligand Immobilization onto Bare Iron Oxide Nanoparticles Enables Efficient Magnetic Separation of Antibodies.

Magnetic separation is a promising alternative to chromatography for enhancing the downstream processing (DSP) of monoclonal antibodies (mAbs). However, there is a lack of efficient magnetic particles for successful application. Aiming to fill this gap, we demonstrate the suitability of bare iron oxide nanoparticles (BION) with physical site-directed immobilization of an engineered Protein A affinity ligand (rSpA) as an innovative magnetic material. The rSpA ligand contains a short peptide tag that enables the direct and stable immobilization onto the uncoated BION surface without commonly required laborious particle activation. The resulting BION@rSpA have beneficial characteristics outperforming conventional Protein A-functionalized magnetic particles: a simple, fast, low-cost synthesis, a particle size in the nanometer range with a large effective specific surface area enabling large immunoglobulin G (IgG) binding capacity, and a high magnetophoretic velocity advantageous for fast processing. We further show rapid interactions of IgG with the easily accessible rSpA ligands. The binding of IgG to BION@rSpA is thereby highly selective and not impeded by impurity molecules in perfusion cell culture supernatant. Regarding the subsequent acidic IgG elution from BION@rSpA@IgG, we observed a hampering pH increase caused by the protonation of large iron oxide surfaces after concentrating the particles in 100 mM sodium acetate buffer. However, the pH can be stabilized by adding 50 mM glycine to the elution buffer, resulting in recoveries above 85% even at high particle concentrations. Our work shows that BION@rSpA enable efficient magnetic mAb separation and could help to overcome emerging bottlenecks in DSP.

Optical biosensing of monkeypox virus using novel recombinant silica-binding proteins for site-directed antibody immobilization

The efficient immobilization of capture antibodies is crucial for timely pathogen detection during global pandemic outbreaks. Therefore, we proposed a silica-binding protein featuring core functional domains (cSP). It comprises a peptide with a silica-binding tag designed to adhere to silica surfaces and tandem protein G fragments (2C2) for effective antibody capture. This innovation facilitates precise site-directed immobilization of antibodies onto silica surfaces. We applied cSP to silica-coated optical fibers, creating a fiber-optic biolayer interferometer (FO-BLI) biosensor capable of monitoring the monkeypox virus (MPXV) protein A29L in spiked clinical samples to rapidly detect the MPXV. The cSP-based FO-BLI biosensor for MPXV demonstrated a limit of detection (LOD) of 0.62 ng/mL in buffer, comparable to the 0.52 ng/mL LOD achieved using a conventional streptavidin (SA)-based FO-BLI biosensor. Furthermore, it achieved LODs of 0.77 ng/mL in spiked serum and 0.80 ng/mL in spiked saliva, exhibiting no cross-reactivity with other viral antigens. The MPXV detection process was completed within 14 min. We further proposed a cSP-based multi-virus biosensor strategy capable of detecting various pandemic strains, such as MPXV, the latest coronavirus disease (COVID) variants, and influenza A protein, to extend its versatility. The proposed cSP-modified FO-BLI biosensor has a high potential for rapidly and accurately detecting MPXV antigens, making valuable contributions to epidemiological studies.

Site-directed immobilization of enzymes on nanoparticles using self-assembly systems

Enzyme immobilization is an effective method for improving the stability and reusability. However, linking at random sites on the enzyme results in low catalytic efficiency due to blockage of the active site or conformational changes. Therefore, controlling the orientation of enzymes on the carrier has been developed. Here, the site-specific mutation and the SpyTag/SpyCatcher systems were used to prepare a site-directed immobilized enzyme. The thermal stability of the immobilized enzyme was better than that of the free enzyme, and ≥80 % of the catalytic activity was retained after 30 days of storage. Furthermore, the Michaelis constant (Km) and the turnover number (kcat) of the immobilized enzyme were 5.23-fold lower and 6.11-fold higher than those of the free enzyme, respectively, which appeared to be related to changes in secondary structure after immobilization. These findings provide a new and effective option for enzyme-directed immobilization.

Site-directed display of zearalenone lactonase on spilt-intein functionalized nanocarrier for green and efficient detoxification of zearalenone

In this study, we prepared a functional organic–inorganic hybrid nanoflower (InHNF) via split intein moiety in a biomineralization process without using organic solvents. InHNF could specifically bind the target enzymes from crude cell lysates within seconds and site-directedly display them on the surface by forming a peptide bond with enzyme’s terminal amino acid residue. This unique feature enabled InHNF to increase the specific activity of zearalenone detoxifying enzyme ZHD518 by 40 ∼ 60% at all tested temperatures and prevented enzyme denaturation even under extreme pH conditions (pH 3–11). Furthermore, it exhibited excellent operational stability, with a residual activity of over 70% after eight reaction cycles. Strikingly, InHNF-ZHD518 achieved above 50% ZEN degradation despite the near inactivation of free ZHD518 in beer sample. Overall, InHNF nanocarriers can achieve environmentally friendly, purification-free, and site-directed immobilization of food enzymes and enhance their catalytic properties, making them suitable for a wide range of industrial applications.

High-affinity peptides developed against calprotectin and their application as synthetic ligands in diagnostic assays

Common inflammatory disorders such as ulcerative colitis and Crohn’s disease are non-invasively diagnosed or monitored by the biomarker calprotectin. However, current quantitative tests for calprotectin are antibody-based and vary depending on the type of antibody and assay used. Additionally, the binding epitopes of applied antibodies are not characterized by structures and for most antibodies it is unclear if they detect calprotectin dimer, tetramer, or both. Herein, we develop calprotectin ligands based on peptides, that offer advantages such as homogenous chemical composition, heat-stability, site-directed immobilization, and chemical synthesis at high purity and at low cost. By screening a 100-billion peptide phage display library against calprotectin, we identified a high-affinity peptide (Kd = 26 ± 3 nM) that binds to a large surface region (951 Å2) as shown by X-ray structure analysis. The peptide uniquely binds the calprotectin tetramer, which enabled robust and sensitive quantification of a defined species of calprotectin by ELISA and lateral flow assays in patient samples, and thus offers an ideal affinity reagent for next-generation inflammatory disease diagnostic assays.

Controlled Immobilization of a Palladium Complex/Laccase Hybrid into a Macrocellular Siliceous Host.

This study investigates the site-directed immobilization of a hybrid catalyst bearing a biquinoline-based-Pd(II) complex (1) and a robust laccase within cavities of a silica foam to favor veratryl alcohol oxidation. We performed the grafting of 1 at a unique surface located lysine of two laccase variants, either at closed (1⊂UNIK157 ) or opposite position (1⊂UNIK71 ) of the enzyme oxidation site. After immobilization into the cavities of silica monoliths bearing hierarchical porosity, we show that catalytic activity is dependent on the orientation and loading of each hybrid, 1⊂UNIK157 being twice as active than 1⊂UNIK71 (203 TON vs 100 TON) when operating under continuous flow. These systems can be reused 5 times, with an operational activity remaining as high as 40 %. We show that the synergy between 1 and laccase can be tuned within the foam. This work is a proof of concept for controlling the organization of a heterogeneous hybrid catalyst using a Pd/laccase/silica foam.

Resorc[4]arene Modifiers for Supramolecular Site-Directed Immobilization of Antibodies on Multi-Walled Carbon Nanotubes.

One of the main problems in developing immunosensors featuring carbon nanotubes (CNTs) is immobilizing antibodies (Abs) onto the CNT surface to afford selective binding to target antigens (Ags). In this work, we developed a practical supramolecular Ab conjugation strategy based on resorc[4]arene modifiers. To improve the Ab orientation on the CNTs surface and optimizing the Ab/Ag interaction, we exploited the host-guest approach by synthesizing two newly resorc[4]arene linkers R1 and R2 via well-established procedures. The upper rim was decorated with eight methoxyl groups to promote selective recognition of the fragment crystallizable (Fc ) region of the Ab. Moreover, the lower rim was functionalized with 3-bromopropyloxy or 3-azidopropiloxy substituents to bind the macrocycles on the multi-walled carbon nanotubes (MWCNTs) surface. Accordingly, several chemical modifications of MWCNTs were evaluated. After the morphological and electrochemical characterization of nanomaterials, the resorc[4]arene-modified MWCNTs were deposited onto a glassy carbon electrode surface to evaluate their potential applicability for label-free immunosensor development. The most promising system showed an improved electrode active area (AEL ) of almost 20 % and a site-oriented immobilization of the SARS-CoV-2 spike protein S1 antibody (Ab-SPS1). The developed immunosensor revealed a good sensitivity (23.64 μA mL ng-1 cm-2 ) towards the SPS1 antigen and a limit of detection (LOD) of 1.01 ng mL-1 .

Surface modification for site-directed covalent attachment of molecules via strain-promoted azide-alkyne click-chemistry reaction and photolithography

Copper-free “click” chemistry on appropriately modified surfaces and photolithography were explored for site-directed immobilization of biomolecules. The surfaces were modified either with self-assembled monolayers of silanes or an epoxy resin and then used as they were or further modified through adsorption of rabbit gamma-globulins to increase their reactive amine-content. All surfaces were then reacted with a succinimidyl ester cyclooctyne derivative and the efficiency of each modification approach was determined through click reaction with an azide derivative of fluorescein. It was found that the highest fluorescence signal was provided by the surface that had been modified with the epoxy resin and then coated with rabbit gamma-globulins. Surfaces prepared following the different modification procedures have been analyzed with ToF-SIMS to gain insight into the chemical changes of the surfaces after each step of the modification procedure. Analysis of intensities of characteristic ions signals confirmed the successful outcome of click reaction following the proposed surface modification approach. In addition, the ability to define through photolithography areas for site-directed immobilization of biomolecules onto surfaces modified with the succinimidyl ester cyclooctyne derivative was demonstrated through the creation of two molecules patterns.

Evolving enzymatic electrochemistry with rare or unnatural amino acids

Proper orientation of oxidoreductases on electrodes is important for efficient electron transfer in bioelectrochemical studies. Site-directed mutagenesis confers the ability to control the orientation of enzymes on electrodes in addition to modifying enzyme catalytic properties and understanding native electron transfer mechanisms and protein–protein interactions. Although numerous improvements have been achieved in the site-directed immobilization of enzymes, they are limited to the use of the 20 “standard” amino acids of the protein. This opinion considers the utilization of unnatural amino acids (UAAs) to introduce unique functional groups to proteins for their site-specific immobilization and subsequent enzymatic electrochemistry studies. Moreover, the importance of the site-specific incorporation of selenocysteine is described due to its potential to improve/alter the electrochemical properties.

The Effect of the Topmost Layer and the Type of Bone Morphogenetic Protein-2 Immobilization on the Mesenchymal Stem Cell Response.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) plays a key role in the stem cell response, not only via its influence on osteogenesis, but also on cellular adhesion, migration, and proliferation. However, when applied clinically, its supra-physiological levels cause many adverse effects. Therefore, there is a need to concomitantly retain the biological activity of BMP-2 and reduce its doses. Currently, the most promising strategies involve site-specific and site-directed immobilization of rhBMP-2. This work investigated the covalent and electrostatic binding of rhBMP-2 to ultrathin-multilayers with chondroitin sulfate (CS) or diazoresin (DR) as the topmost layer. Angle-resolved X-ray photoelectron spectroscopy was used to study the exposed chemical groups. The rhBMP-2 binding efficiency and protein state were studied with time-of-flight secondary ion mass spectrometry. Quartz crystal microbalance, atomic force microscopy, and enzyme-linked immunosorbent assay were used to analyze protein–substrate interactions. The effect of the topmost layer was tested on initial cell adhesion and short-term osteogenesis marker expression. The results show the highest expression of selected osteomarkers in cells cultured on the DR-ended layer, while the cellular flattening was rather poor compared to the CS-ended system. rhBMP-2 adhesion was observed only on negatively charged layers. Cell flattening became more prominent in the presence of the protein, even though the osteogenic gene expression decreased.

Comparison of Random and Site-Directed Immobilization of Antibody for α-Fetoprotein Direct Immunoassay by Protein Chip

For immunoassay, immobilization of antibody is the most important technique in bioengineering for maintaining its intrinsic activity of antigen binding. The sugar chain in the Fc region carried by antibody can be oxidized into aldehyde group by sodium m-periodate, which can couple with the amino-group on chemistry modified substrate surface through covalent linking. Thereby, in situ site-directed immobilization of the oxidized antibody for only one-step reaction can be performed. Here we propose a facile method that oxidized anti-human α-fetoprotein antibody (anti-AFP) was in situ site-directed immobilized on APTES treated silicon substrate utilizing microfluidic channel system for AFP immunoassay. The shape of the immobilized anti-AFP was observed by AFM and imaged by imaging ellipsometry of self-developed protein system. Results shown that the amount of antibody immobilization amount and capacity of antigen binding of random immobilization and site-directed immobilization were separately enhanced 16.0% and 7.0%. the suggested strategy has the advantages of simple, environment-friend and convenient-operation.

Site-Directed Immobilization of an Engineered Bone Morphogenetic Protein 2 (BMP2) Variant to Collagen-Based Microspheres Induces Bone Formation In Vivo.

For the treatment of large bone defects, the commonly used technique of autologous bone grafting presents several drawbacks and limitations. With the discovery of the bone-inducing capabilities of bone morphogenetic protein 2 (BMP2), several delivery techniques were developed and translated to clinical applications. Implantation of scaffolds containing adsorbed BMP2 showed promising results. However, off-label use of this protein-scaffold combination caused severe complications due to an uncontrolled release of the growth factor, which has to be applied in supraphysiological doses in order to induce bone formation. Here, we propose an alternative strategy that focuses on the covalent immobilization of an engineered BMP2 variant to biocompatible scaffolds. The new BMP2 variant harbors an artificial amino acid with a specific functional group, allowing a site-directed covalent scaffold functionalization. The introduced artificial amino acid does not alter BMP2′s bioactivity in vitro. When applied in vivo, the covalently coupled BMP2 variant induces the formation of bone tissue characterized by a structurally different morphology compared to that induced by the same scaffold containing ab-/adsorbed wild-type BMP2. Our results clearly show that this innovative technique comprises translational potential for the development of novel osteoinductive materials, improving safety for patients and reducing costs.

Magnetic Separation of Antibodies with High Binding Capacity by Site-Directed Immobilization of Protein A-Domains to Bare Iron Oxide Nanoparticles

The demand for purified antibodies is ever-rising. This study presents a nanoparticle-based material for efficient magnetic separation of immunoglobulin G (IgG) with high binding capacity. The char...

Controlling Conjugated Antibodies at the Molecular Level for Active Targeting Nanoparticles toward HER2-Positive Cancer Cells.

For active targeting nanodrug delivery systems conjugated with antibodies, both lack of control of the antibody at the molecular level and protein corona formation remarkably decreases targeting efficacy. Herein, we designed a series of silica nanoparticles toward HER2-positive breast cancer cells, with an anti-HER2 Fab-6His density ranging from 50 to 180 molecules per nanoparticle. Through the site-directed immobilization method we developed, the antigen-binding domain of anti-HER2 Fab was mostly accessible to the HER2 receptor. Both polyethylene glycol (PEG) chains and a high density of Fab were shown to suppress protein corona formation and macrophage uptake. The dependency of targeting efficacy and cytotoxicity on Fab density was shown using a series of breast cancer cell lines with different levels of the HER2 expression. The high density of Fab stimulates quick responses from HER2-positive cells. Combined with PEG chains, conjugated antibodies with a well-controlled orientation and density significantly improves delivery performance and sheds light on the design and preparation of an improved active targeting nanodrug delivery system.

Protein A-based ligands for affinity chromatography of antibodies

Protein A chromatography is a key technology in the industrial production of antibodies, and a variety of commercial protein A adsorbents are available in shelf. High stability and binding capacity of a protein A adsorbent are two key issues for successful practice of protein A chromatography. Earlier versions of protein A adsorbents ever exhibited serious fragility to typical cleaning-in-place protocols (e.g. washing with sodium hydroxide solution), and suffered from low binding capacity, harsh elution, ligand leakage and other problems involved in industrial applications. During the last three decades, various techniques and approaches have been applied in the improvement of chemical stability and enhancement of binding capacity of protein A-based ligands and adsorbents for antibody purifications. This mini-review focuses on the technical explorations in protein A-based affinity adsorbents, especially protein A-based ligands, including the efforts to increase the chemical stability by site-directed mutations and to improve the binding capacity by ligand polymerization and site-directed immobilization. Moreover, the efforts to develop short peptide ligands based on the structure of protein A, including the biomimetic design strategies and the synthesis of peptide-mixed mode hybrid ligands are discussed. These peptide and peptide-based hybrid ligands exhibit high affinity and selectivity to antibodies, but noteworthy differences in the binding mechanism of antibody from protein A. As a result, bound antibody to the ligands could be effectively eluted under mild conditions. Perspectives for the development of the protein A-based peptide ligands have been extensively discussed, suggesting that the ligands represent a direction for technological development of antibody purification.

Design and site-directed immobilization of single-chain Fv antibody to polystyrene latex beads via material-binding peptides and application to latex turbidimetric assay

In this study, immobilization of single-chain Fv (scFv) antibodies on the surfaces of polystyrene (PS) latex beads via material-binding peptides was investigated for sensitive immuno-turbidimetric assay of C-reactive protein (CRP). Anti-CRP scFvs fused with polystyrene-binding peptide (PS-tag) and poly(methylmethacrylate)-binding peptide (PMMA-tag) were over-expressed in Escherichiacoli cells and recovered in the active form following refolding. The beads with PMMA-tag-fused scFv (scFv-PM) were successfully suspended with sufficient dispersion at pH 8.0. Three types of alternative scFv-PMs with a penta-asparatic acid tag (D5-tag) introduced at different positions were then designed. All of the D5-tagged scFv-PMs were successfully immobilized on the surfaces of beads with no significant change in the diameter of the latex beads at pH levels ranging from 6.0 to 8.0. According to the results of turbidimetric assay for the detection of CRP, 13ng/ml of CRP was detectable using beads with D5-tagged scFv-PMs at 400ng/cm3, and no turbidity change was observed in the absence of antigen. When the density of scFv-PM was 250ng/cm2, which was 63% of the maximum density, the beads were dispersed well and reactive with the antigen at a concentration range comparable to those with D5-tagged scFv-PMs. These results indicate that controlling charge density on the surface of beads after site-directed immobilization is definitely important in order to maintain high levels of dispersion and reactivity. Thus, the usefulness of the scFv-PM as well as D5-tagged scFv-PMs developed in the present study should be significant when used as ligand antibodies in the preparation of immuno-latex beads.

Construction of Immunomagnetic Particles with High Stability in Stringent Conditions by Site-Directed Immobilization of Multivalent Nanobodies onto Bacterial Magnetic Particles for the Environmental Detection of Tetrabromobisphenol-A.

Bacterial magnetic particles (BMPs) are an attractive carrier material for immunoassays because of their nanoscale size, dispersal ability, and membrane-bound structure. Antitetrabromobisphenol-A (TBBPA) nanobodies (Nbs) in the form of monovalence (Nb1), bivalence (Nb2), and trivalence (Nb3) were biotinylated and immobilized onto streptavidin (SA)-derivatized BMPs to construct the complexes of BMP-SA-Biotin-Nb1, -Nb2, and -Nb3, respectively. An increasing order of binding capability of BMP-SA-Biotin-Nb1, -Nb2, and -Nb3 to TBBPA was observed. These complexes showed high resilience to temperature (90 °C), methanol (100%), high pH (12), and strong ionic strength (1.37 M NaCl). A BMP-SA-Biotin-Nb3-based enzyme linked immunosorbent assay (ELISA) for TBBPA dissolved in methanol was developed, showing a half-maximum inhibition concentration (IC50) of 0.42 ng mL-1. TBBPA residues in landfill leachate, sewage, and sludge samples determined by this assay were in a range of <LOD-1.17 ng mL-1, <LOD-0.75 ng mL-1, and <LOD-3.65 ng g-1 (dw), respectively, correlating well with the results by liquid chromatography tandem mass spectrometry. The BMP-SA-Biotin-Nb3 was reusable at least three times without significant loss of the binding capability. The BMP-SA-Biotin-Nb3-based ELISA, with a total assay time of less than 30 min, is promising for the rapid monitoring of TBBPA in the environment.

The capture of antibodies by antibody-binding proteins for ABO blood typing using SPR imaging-based sensing technology

In this study, a wavelength-interrogated surface plasmon resonance imaging-based immunosensor was developed for ABO blood typing. To construct the immunosensor, the detection line array was formed by the site-directed immobilization of blood group antibodies on SPR chip modified with antibody-binding proteins. Red blood cell (RBC) samples were flowed into the multichannel flow cell that was orthogonal to the detection line arrays. By serially analyzing the interaction between RBCs, antibody A, and antibody B through the observed changes in the resonance dip, blood typing of four RBC samples was achieved in a single run. Detailed investigations focused on optimizing the conditions that affected the efficiency and sensitivity of immunoassay. Under the optimized conditions, the RBC samples were appropriately grouped in less than 1 h, including chip modification time. The lowest detection limit for RBC-A and RBC-B was 2×106 cells/ml. The immunosensor developed through this study provided a simple, rapid, high-throughput, and sensitive immunology strategy for classification of ABO blood group.