Purification of anti-chymotrypsin antibodies for the preparation of a bioaffinity matrix with oriented chymotrypsin as immobilized ligand

Polyclonal antibodies suitable for the oriented immobilization of chymotrypsin we prepared by chromatography on a bioaffinity matrix which had the enzyme immobilized through its active site to antilysin, covalently linked to bead cellulose. After periodate oxidation of their carbohydrate moieties, the isolated antibodies were coupled to a hydrazide derivative of bead cellulose. The periodate oxidation step, which led to greater efficiency and stability of the immunosorbent, had no deleterious effect on antibody activity as assessed by ELISA. Addition of chymotrypsin to the immunosorbent yielded an enzymically active bioaffinity matrix with the optimum molar enzyme/antibody ratio of 2.     

Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.     

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments

Factors affecting the specific activity of immobilized antibodies and their biologically active fragments were studied with goat anti-mouse and goat anti-human immunoglobulin G. Antibodies were immobilized on HW 65 polymeric support matrix activated with carbonyldiimidazole, hydrazide and iodoacetic acid. The most significant factors influencing the specific activity of stochastic coupling of antibodies are multisite attachment, multiple orientations and steric hindrance imposed by crowding of antibody and the size of the antigen. In oriented immobilization the specific activity is affected only by steric hindrance. The specific activity of immunosorbents prepared by immobilization of F(ab′) fragments can be improved to almost 100% by limiting the amount of protein immobilization and the size of the antigen. The present study shows the protocols for optimizing immobilized antibody performance.     

Fabrication of an antibody microwell array with self-adhering antibody binding protein

One of the promising methods of preparing antibody arrays is immobilizing antibodies with protein A or protein G, each of which binds specifically to the heavy chain constant (Fc) region of immunoglobulin G (IgG). In this system, antibody immobilization efficiency depends on the number of active Fc binding proteins that need to be immobilized on the surface. Here we have designed and constructed an Fc binding protein with a self-adhering ability that can be immobilized on the hydrophobic surface by simple adsorption. It consists of an Fc binding domain of protein G (G3) and hydrophobic domain of elastin (E72). Direct observation revealed its self-adhering ability on the hydrophobic surface. The enzyme-linked immunosorbent assay (ELISA) showed that it retained antibody binding ability on the surface. The antibody array model was prepared on a hydrophobic microwell glass slide with E72G3, which specifically detect the antigen with a sevenfold greater sensitivity than the G3-treated slide. These results suggest that the E72G3 is useful for simple and effective immobilization of antibodies and can be used to fabricate any immuno devices.     

Versatile method for production and controlled polymer-immobilization of biologically active recombinant proteins

The immobilization of a protein by covalent attachment to a support matrix should involve only functional groups of the protein that are not essential for its biological activity. A general strategy for obtaining recombinant proteins designed for oriented covalent grafting onto copolymers was investigated. The rationale involves the definition of seven p24-derived recombinant proteins as fused to either distant or adjacent tags comprising primary amine rich tag consisting of six contiguous lysines suitable for oriented covalent immobilization and a hexa-histidine tag suitable for metal chelate affinity purification. High-level expression, efficient affinity purification, and coupling yields onto maleic anhydride-alt-methyl vinyl ether copolymers higher than 95% were obtained for all proteins. Afterwards, an investigation of the biological features of the immobilized vs. nonimmobilized protein onto the copolymer allowed us to select one bioconjugate which was used in a diagnostic context, i.e., as a capture antigen in an ELISA format test. Sera from 107 HIV-seropositive individuals at various stages of HIV infection, including two seroconversion panels and 104 healthy HIV-seronegative controls, were tested using either RH24 or RK24H-copolymer coated onto the microtiter plate. These assays showed that the use of such a protein-copolymer bioconjugate allowed detection of lower antibody titers than the RH24 protein, illustrating the potential of applications of such doubly tagged proteins. Thus, a set of expression vectors was designed containing four different combinations of hexa-lysine and hexa-histidine tags and a multiple cloning site, allowing the production of different recombinant fusion proteins suitable for biological reactivity conservation after immobilization.     

Use of dextrans as long and hydrophilic spacer arms to improve the performance of immobilized proteins acting on macromolecules

New dextran-agarose supports, suitable for covalent immobilization of enzymes and proteins acting on macromolecular substrates, were prepared. The thick internal fibers of agarose gels were covered by a low-density layer of long, flexible, hydrophilic, and inert dextran molecules. Rennin and protein A were immobilized on these novel supports and the resulting derivatives exhibited a very high capacity for biological recognition of soluble macromolecular substrates. Caseinolytic activity of this immobilized enzyme was 15-fold higher than activity of directly immobilized rennin, through short spacer arms, on agarose gels. Similarly, the new derivatives of immobilized protein A were able to adsorb up to 2 molecules of immunoglobulin per each molecule of immobilized protein A. When the immobilized proteins were secluded away from the support surface by using these new long and hydrophilic spacer arms, they exhibit minimal steric hindrances that could be promoted by the proximity of the support surface.     

Essential role of the concentration of immobilized ligands in affinity chromatography:: Purification of guanidinobenzoatase on an ionized ligand

Guanidinobenzoatase, a plasma protein with possible application as a ‘tumor marker’, has been fully purified by one-step affinity chromatography. The affinity matrix was prepared by ‘controlled’ immobilization of an enzyme inhibitor (agmatine) onto commercial agarose gels containing carboxyl moieties activated as N-hydroxysuccinimide esters. In this way, agmatine becomes immobilized through an amido bond and preserves an ionized guanidino moiety. Different matrices with different concentration of ligands were prepared in order to evaluate their properties as affinity supports. Interestingly, matrices with a very low concentration of immobilized ligands (2 μmol/ml, corresponding to the modification of only 5% of active groups in the commercial resins) exhibited a low capacity for unspecific adsorption of proteins (as anion-exchange resins) and displayed also a high capacity for specific adsorption of our target protein. On the other hand, when affinity matrices possessed a moderate concentration of agmatine (10 μmol/ml of gel or higher), two undesirable phenomena were observed: (a) the matrix behaves as a very good anionic exchange support able to non-specifically adsorb most of plasma proteins and (b) the specific adsorption of our target protein becomes much lower. The latter phenomenon could be due to steric hindrances promoted by the interaction between each individual immobilized ligand and the corresponding binding pocket in the target protein. These hindrances could also be promoted by the presence of a fairly dense layer of immobilized ligands covering the support surface, thus preventing interactions between immobilized ligands and partially buried protein-binding pockets. In this way, a successful affinity purification (23.5% yield, ×220 purification factor, a unique electrophoretic band) could be achieved by combination of three approaches: (i) the use of affinity matrices possessing a very low density of immobilized ligands, (ii) performing affinity adsorption at high ionic strength and (iii) performing specific desorption with substrates or substrate analogues.     

Atomic force spectroscopy-based study of antibody pesticide interactions for characterization of immunosensor surface

Development of immunobiosensor detector surfaces involves the immobilization of active antibodies on the capture surface without any significant loss of antigen binding activity. An atomic force microscope (AFM) was used to directly evaluate specific interactions between pesticides and antibodies on a biosensor surface. Oriented immobilization of antibodies against two herbicide molecules 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine, on gold, was carried out to create the active immunobiosensor surfaces. The adhesive forces between immobilized antibodies and their respective antigens were measured by force spectroscopy using hapten-carrier protein functionalized AFM cantilevers. Relative functional affinity (avidity) measurements of the antibodies carried out prior to immobilization, well correlated with subsequent AFM force measurement observations. Analysis showed that immobilization had not compromised the reactivity of the surface immobilized antibody molecules for antigen nor was there any change in their relative quality with respect to each other. The utility of the immunoreactive surface was further confirmed using a Surface Plasmon Resonance (SPR) based detection system. Our study indicates that AFM can be utilized as a convenient immunobiosensing tool for confirming the presence and also assessing the strength of antibody-hapten interactions on biosensor surfaces under development.     

蛋白质微阵列检测抗原-抗体相互作用

为了制备蛋白质微阵列和研究芯片表面抗原-抗体的相互作用,研究了如何在玻片表面固化蛋白质和用荧光染料（Cy3,Cy5）对蛋白质进行标记.结果表明,在醛基修饰的玻璃表面,通过共价偶联的方法将抗原或抗体固定到芯片表面,能使二者保持其特异性结合能力.同时,荧光标记后的抗原或抗体仍然具有特异性结合能力.蛋白质微阵列是通过机械手在玻片表面排阵制作的.芯片上的荧光信号获取采用了激光共焦荧光扫描系统.用不同浓度的抗原探针阵列,对其相应的抗体靶分子的特异性结合进行了分析和研究.此外,还通过在玻片表面固定兔IgG和固定鼠IgG,对羊抗兔和羊抗鼠抗体与其相应抗原的特异性相互作用进行了检测.     

An artificial processivity clamp made with streptavidin facilitates oriented attachment of polymerase-DNA complexes to surfaces

Single molecule analysis of individual enzymes can require oriented immobilization of the subject molecules on a detection surface. As part of a technology development project for single molecule DNA sequencing, we faced the multiple challenges of immobilizing both a DNA polymerase and its DNA template together in an active, stable complex capable of highly processive DNA synthesis on a nonstick surface. Here, we report the genetic modification of the archaeal DNA polymerase 9 degrees N in which two biotinylated peptide 'legs' are inserted at positions flanking the DNA-binding cleft. Streptavidin binding on either side of the cleft both traps the DNA template in the polymerase and orients the complex on a biotinylated surface. We present evidence that purified polymerase-DNA-streptavidin complexes are active both in solution and immobilized on a surface. Processivity is improved from <20 nt in the unmodified polymerase to several thousand nucleotides in the engineered complexes. High-molecular weight DNA synthesized by immobilized complexes is observed moving above the surface even as it remains tethered to the polymerase. Pre-formed polymerase-DNA-streptavidin complexes can be stored frozen and subsequently thawed without dissociation or loss of activity, making them convenient for use in single molecule analysis.     

Preparing a highly specific inert immunomolecular-magnetic beads for rapid detection and separation of <Emphasis Type="Italic">S. aureus</Emphasis> and group G <Emphasis Type="Italic">Streptococcus</Emphasis>

The rapid detection and separation of Staphylococcus aureus and group G Streptococcus was based on the affinity chromatography interactions between Fc fragment of human IgG and protein A/G (located on the cell wall of S. aureus and group G Streptococcus). In this case, immobilization of antibodies had to take place in a different and complementary way than in the case of conventional immunosensors. In this study, three different kinds of immunomolecular-magnetic beads (IMB) were prepared for rapid detection and separation of S. aureus and group G Streptococcus (GGS). The Fc regions of the immobilized antibodies were fully accessible to adsorb protein A or protein G. On the contrary, conventional immunosensors had to have fully accessible Fab regions to facilitate the antigen–antibody recognition. It was suggested that the worse method of immobilization of the antibodies for conventional use would yield the better results for this specific use. In this study, we also perfectly solved the nonspecific adsorptions and interaction problems, which were the most serious critical problems for all kinds of sensors. It was achieved by blocking the excess surface groups of aldehyde IMB and the Fab region of the immobilized antibodies with aldehyde-dextran.     

Binding and signaling of surface-immobilized reagentless fluorescent biosensors derived from periplasmic binding proteins

Development of biosensor devices typically requires incorporation of the molecular recognition element into a solid surface for interfacing with a signal detector. One approach is to immobilize the signal transducing protein directly on a solid surface. Here we compare the effects of two direct immobilization methods on ligand binding, kinetics, and signal transduction of reagentless fluorescent biosensors based on engineered periplasmic binding proteins. We used thermostable ribose and glucose binding proteins cloned from Thermoanaerobacter tengcongensis and Thermotoga maritima, respectively. To test the behavior of these proteins in semispecifically oriented layers, we covalently modified lysine residues with biotin or sulfhydryl functions, and attached the conjugates to plastic surfaces derivatized with streptavidin or maleimide, respectively. The immobilized proteins retained ligand binding and signal transduction but with adversely affected affinities and signal amplitudes for the thiolated, but not the biotinylated, proteins. We also immobilized these proteins in a more specifically oriented layer to maleimide-derivatized plates using a His(2)Cys(2) zinc finger domain fused at either their N or C termini. Proteins immobilized this way either retained, or displayed enhanced, ligand affinity and signal amplitude. In all cases tested ligand binding by immobilized proteins is reversible, as demonstrated by several iterations of ligand loading and elution. The kinetics of ligand exchange with the immobilized proteins are on the order of seconds.     

Silver ion microplates for immunoassays.

Microplate wells can be coated with silver ions using glutaraldehyde as a spacer molecule and thiourea as a complexing ligand. Microwells containing surface silver ions are shown to immobilize biotin-labeled horseradish peroxidase (HRP) in active form, while showing very little affinity for the unlabeled enzyme. These plates can also immobilize biotin-labeled antibodies that exhibit bioactivity after immobilization. Silver ions are needed for the complexation of the biotinylated enzyme or antibody because microwells modified to contain surface amine or thiourea molecules do not immobilize appreciable amounts of the labeled proteins. A maximum surface coverage for biotin-labeled HRP of 40 ng/cm2 and an immobilization binding constant of Km = 8 x 10(9)/M are determined from serial dilutions in a microplate. Detection of as little as 6.7 fmol HRP is achieved using antibodies immobilized on the silver ion-modified microplates. Active antibody surface densities were estimated to be between 130 and 260 nm2/antibody molecule. Background binding of HRP to the modified silver ion microplates was very low, allowing for reasonably accurate detection between 10(-14) and 10(-11) mol HRP.     

Increase in conformational stability of enzymes immobilized on epoxy-activated supports by favoring additional multipoint covalent attachment*

Epoxy supports (Eupergit C) may be very suitable to achieve the multipoint covalent attachment of proteins and enzymes, therefore, to stabilize their three-dimensional structure. To achieve a significant multipoint covalent attachment, the control of the experimental conditions was found to be critical. A three-step immobilization/stabilization procedure is here proposed: 1) the enzyme is firstly covalently immobilized under very mild experimental conditions (e.g. pH 7.0 and 20 degrees C); 2) the already immobilized enzyme is further incubated under more drastic conditions (higher pH values, longer incubation periods, etc.) to "facilitate" the formation of new covalent linkages between the immobilized enzyme molecule and the support; 3) the remaining groups of the support are blocked to stop any additional interaction between the enzyme and the support. Progressive establishment of new enzyme-support attachments was showed by the progressive irreversible covalent immobilization of several subunits of multi-subunits proteins (all non-covalent structures contained in crude extracts of different microorganism, penicillin G acylase and chymotrypsin). This multipoint covalent attachment enabled the significant thermostabilization of two relevant enzymes, (compared with the just immobilized derivatives): chymotrypsin (5-fold factor) and penicillin G acylase (18-fold factor). Bearing in mind that this stabilization was additive to that achieved by conventional immobilization, the final stabilization factor become 100-fold comparing soluble penicillin G acylase and optimal derivative. These stabilizations were observed also when the inactivations were promoted by the enzyme exposure to drastic pH values or the presence of cosolvents.     

Affinity immobilization of a genetically engineered bifunctional hybrid protein.

Protein A from Staphylococcus aureus (SpA) is a receptor for the Fc domain of several classes of antibodies including immunoglobin G (IgG). A hybrid protein consisting of protein A and the enzyme beta-lactamase has been constructed using recombinant DNA techniques. The functional characteristics of the hybrid protein adsorbed on IgG-coated Sepharose matrices were studied in detail and compared to those of (i) the hybrid protein in solution and (ii) beta-lactamase covalently immobilized on CNBr-activated Sepharose. Protein A--beta-lactamase bound tightly and specifically to IgG-Sepharose and could be stored for at least 4 weeks without dissociation. The rate of penicillin G hydrolysis by the beta-lactamase domain of the immobilized hybrid protein was found to depend on the amount of IgG covalently coupled to the support. For all IgG loads, higher specific activities and lower Km values relative to covalently immobilized beta-lactamase were obtained. Adsorption of the hybrid protein on the support resulted in increased stability to thermal deactivation. These results indicate that bifunctional hybrid proteins can be useful for the affinity immobilization of enzymes.     

Mapping HA‐tagged protein at the surface of living cells by atomic force microscopy

Single‐molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2‐adrenergic receptor (β2‐AR), a G protein‐coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2‐AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.     

Recent advances in nano-carrier immobilized enzymes and their applications

Enzymes have been widely used because of their catalytic properties, and immobilization is a promising technique to improve their catalytic activity and stability. Due to their large specific surface areas, exceptional chemical, mechanical, thermal and cost effective characteristics, nanomaterials should be ideal carriers for the immobilization of enzymes. Enzymes immobilized on nano-carriers are more robust and stable, and can be recycled and reused. This review focuses on the nanomaterial immobilized enzymes and their applications. The introduction addresses the advantages of immobilized enzymes and the features of enzyme immobilization nanocarriers. The next section covers carbonaceous nanomaterials used in enzymes immobilization, with subsections on carbon nanotube, graphene, graphene oxide and reduced graphene oxide. The third section treats metallic nanomaterials for enzymes immobilization, with subsections on metal (gold), metal oxide (titanium dioxide, zinc oxide) and metal hydroxide (layered double hydroxide) nanomaterials. Then, the next section summarizes the applications of nanomaterial immobilized enzymes. A concluding section discusses the challenges and prospects of nanomaterial immobilized enzymes.     

Protected immobilization of Taq DNA polymerase by active site masking on self-assembled monolayers of ω-functionalized thiols

A new efficient immobilization method that enables oriented immobilization of biologically active proteins was developed based on concepts of active site masking and kinetic control. Taq DNA polymerase was immobilized covalently on mixed self-assembled monolayers (SAMs) of ω-carboxylated thiol and ω-hydroxylated thiol through amide bonds between the protein and the carboxyl group in SAMs. Activity of the immobilized enzyme as large as 70% of solution-phase enzyme was achieved by masking the active site of the Taq DNA polymerase prior to the immobilization. In addition, the number of immobilization bonds was controlled by optimizing the carboxyl group concentration in the mixed monolayer. The maximum activity of immobilized Taq DNA polymerase was achieved at 5% of 12-mercaptododecanoic acid. The activity observed with protected immobilized enzyme was approximately 20 times higher than that observed with randomly immobilized enzyme. The maximum activity was acquired at a 1:1 DNA/enzyme masking ratio, immobilization pH 8.3, and within 10 min of reaction time. This concept of the active site masking and kinetic control of the number of covalent bonds between proteins and the surface can be generally applicable to a broad range of proteins to be immobilized on the solid surface with higher activity.     

酶的定向固定化方法及其对酶生物活性的影响

固定化酶可以提高酶的稳定性,但通常酶通过酶分子上的多个赖氨酸残基随意固定在载体上,这样会使酶的活性显著下降,采用定向固定化酶不仅可以提高酶的稳定性,而且可以保存它的活性。综述了定向固定化酶的几种方法,比较了定向固定化和随意固定化对酶活性的影响。另外,还叙述了酶的活性位点结构变化的自旋共振波谱(EPR)检测。     

Oriented immobilization of proteins on hydroxyapatite surface using bifunctional bisphosphonates as linkers

Oriented immobilization of proteins is an important step in creating protein-based functional materials. In this study, a method was developed to orient proteins on hydroxyapatite (HA) surfaces, a widely used bone implant material, to improve protein bioactivity by employing enhanced green fluorescent protein (EGFP) and β-lactamase as model proteins. These proteins have a serine or threonine at their N-terminus that was oxidized with periodate to obtain a single aldehyde group at the same location, which can be used for the site-specific immobilization of the protein. The HA surface was modified with bifunctional hydrazine bisphosphonates (HBPs) of various length and lipophilicity. The number of functional groups on the HBP-modified HA surface, determined by a 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, was found to be 2.8 × 10(-5) mol/mg of HA and unaffected by the length of HBPs. The oxidized proteins were immobilized on the HBP-modified HA surface in an oriented manner through formation of a hydrazone bond. The relative protein immobilization amounts through various HBPs were determined by fluorescence and bicinchoninic acid (BCA) assay and showed no significant effect by length and lipophilicity of HBPs. The relative amount of HBP-immobilized EGFP was found to be 10-15 fold that of adsorbed EGFP, whereas the relative amount of β-lactamase immobilized through HBPs (2, 3, 4, 6, and 7) was not significantly different than adsorbed β-lactamase. The enzymatic activity of HBP-immobilized β-lactamase was measured with cefazolin as substrate, and it was found that the catalytic efficiency of HBP-immobilized β-lactamase improved 2-5 fold over adsorbed β-lactamase. The results obtained demonstrate the feasibility of our oriented immobilization approach and showed an increased activity of the oriented proteins in comparison with adsorbed proteins on the same hydroxyapatite surface matrix.