Improving the activity of immobilized subtilisin by site-directed attachment through a genetically engineered affinity tag

An octapeptide affinity tag, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (temied FLAG), was genetically fused to the C-terminus of subtilisin BPN' (SBT) from Bacillus amyloliquefaciens. The fusion protein SBT-FLAG was immobilized to nonporous polystyrene and silica beads both in a site-directed and a random fashion. Site-directed immobilization was achieved by employing the interaction between protein A and a monoclonal antibody specific for the FLAG peptide, while random immobilization was obtained by using glutaraldehyde as a cross-linking reagent. The activity of the immobilized enzymes was compared. It was found that the site-directed subtilisin had higher catalytic efficiency, kcat/KM, which was more than 7-fold of that of the randomly immobilized enzyme. It was also noted that the site-directly immobilized enzyme had superior storage stability over the homogeneous enzyme.     

Evaluation of Binding Between Electroactive Biotin Derivative and Streptavidin Immobilized on Chitin Film

Evaluation of the streptavidin‐biotin binding at the surface of chitin film was carried out with voltammetry. Immobilization of streptavidin was attempted to the protonated chitin film, based on an electrostatic interaction that hardly causes any change in the protein structure. The streptavidin‐biotin binding was estimated from changes in the electrode response of biotin labeled with an electroactive compound. Although the response of daunomycin as an electroactive compound did not change at an electrode covered with streptavidin/chitin film, the response of the labeled biotin decreased. This observation shows that streptavidin is immobilized on the chitin film and the biotin binds with immobilized streptavidin. Consequently, it was clear that the chitin film is useful as a reaction field for protein‐ligand binding. Generally, a binding event between protein and its ligand in the living body occurs on the cell surface. The electrochemical evaluation of protein‐ligand binding on a natural polysaccharide like chitin membrane surface is important.     

Improving the activity of immobilized subtilisin by site-directed attachment through a genetically engineered affinity tag

An octapeptide affinity tag, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (termed FLAG), was genetically fused to the C-terminus of subtilisin BPN′ (SBT) from Bacillus amyloliquefaciens. The fusion protein SBT-FLAG was immobilized to nonporous polystyrene and silica beads both in a site-directed and a random fashion. Site-directed immobilization was achieved by employing the interaction between protein A and a monoclonal antibody specific for the FLAG peptide, while random immobilization was obtained by using glutaraldehyde as a cross-linking reagent. The activity of the immobilized enzymes was compared. It was found that the site-directed subtilisin had higher catalytic efficiency, k_cat/K_M, which was more than 7-fold of that of the randomly immobilized enzyme. It was also noted that the site-directly immobilized enzyme had superior storage stability over the homogeneous enzyme.     

Non-isotopic receptor assay for benzodiazepines using a biotin-labeled ligand and biotin-immobilized microtiter plate.

A non-isotopic receptor assay for benzodiazepine drugs was developed using a biotin-labeled ligand, biotin-1012S. Biotinylated bovine serum albumin (biotin-BSA) was immobilized onto the wall of microtiter plate wells by simple adsorption. Avidin peroxidase conjugate could be extracted from solution owing to its strong interaction with biotin. The amount of avidin peroxidase taken up on the wall was then determined by measuring the enzyme activity. The competition between immobilized biotin on the wall and free biotin for avidin provided the basis for a solid-phase avidin-biotin binding assay. By this binding assay, not only biotin but also biotin-1012S could be measured sensitively. Because 1012S is a ligand with high affinity to benzodiazepine receptors, biotin-1012S could be utilized as a probe ligand for a non-isotopic receptor assay. Based upon the competition between biotin-1012S and various benzodiazepine drugs for the receptor binding sites, a non-isotopic receptor assay was demonstrated.     

Adsorption and activity of a domoic acid binding antibody fragment on mesoporous silicates

The adsorption of an anti-domoic acid single-chain Fv (scFv) antibody fragment onto a range of mesoporous silicate supports was investigated. The scFv fragment adsorbed to all materials investigated, and pI had an apparently large effect on coating, with the greatest-and fastest-adsorption found on the most negatively charged silicates. Maximal coating levels attainable did not reflect the pore diameters of the materials. The immobilized antibody was functional on all materials and bound its antigen, a naturally occurring neurotoxin produced by shellfish, in a rapidly saturating manner that suggested the antibody adsorbed in a multilayer on the mesoporous particles. The antigen:antibody ratio decreased from 1:1.3 to <1:10 with increasing concentration of immobilized antibody, and the immobilized scFv exhibited no detectable reduction in domoic acid binding over a 42-day incubation period.     

Biological sample analysis with immunoaffinity solid-phase microextraction

A theophylline antiserum was covalently immobilized on the surface of a fused silica fiber, modified with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde, and used as a selective and sensitive extraction medium for the immunoaffinity solid-phase microextraction (SPME) determination of theophylline in serum samples. The specificity of the immunoaffinity SPME fiber was first investigated using a fixed concentration of [3H]theophylline together with various amounts of interference, possessing no cross-reactivity with the theophylline antibody. No significant non-specific binding was observed. The reproducibility of the fiber preparation and the immunoaffinity SPME analysis was also investigated, resulting in a relative standard deviation of 6.1% for five analyses of the same fiber. The antigen-antibody binding isotherm was obtained by analyzing theophylline standards of various concentrations (0.1-5 ng mL(-1)) until saturation values were reached. Initial binding of theophylline was linear with a r2 = 0.968. The cross-reactivity of the theophylline immunoaffinity SPME fiber for the structural analog caffeine was investigated by adding various amounts of caffeine in the presence of theophylline at a saturation concentration and produced a low cross-reactivity value of 0.1%. Finally. spiked serum samples (10 and 50 ng mL(-1)) were successfully analyzed with an excellent correlation with the standard binding isotherm, thus confirming the performance of the immunoaffinity SPME coating for improved bioanalysis.     

Intein-mediated biotinylation of proteins and its application in a protein microarray

We report here the first example using an intein-mediated expression system to generate biotinylated proteins suitable for immobilization onto avidin-functionalized glass slides. With this novel array, proteins are site-specifically immobilized on the glass surface and are able to retain their native activity. The advantage of the avidin/biotin linkage over his-tag/Ni-NTA strategies for protein immobilization is highlighted by its ability to withstand a variety of chemical conditions, which makes this new protein array compatible with most biological assays.     

Porous ceramic bed supports for fused silica packed capillary columns used in liquid chromatography

Porous ceramic bed supports for fused silica packed capillary columns utilized in liquid chromatography were prepared by polymerizing solutions containing potassium silicate in-situ within a column to create a mechanically stable, rugged, and easily constructed termination. The effect of the bed support length on efficiency, and comparisons to glass wool bed supports, were considered in terms of column efficiencies and hydrodynamic variables. Results obtained indicate better performance for the ceramic bed support.     

Site‐oriented immobilization of fusion antigen directed by an affinity ligand,and its validation in an immunoassay

Immobilizing proteins on a solid surface in a site‐specific orientation and maintaining their bioactivity are crucial to the construction of high‐performance immunoassays. In this study, an affinity ligand for polystyrene (PS) surface screened from a phage display peptide library, named Lig1, was genetically fused to the N/C‐terminus of chimeric antigen HCV that could be recognized by specific antibodies against hepatitis C virus (HCV). Immunoassay characteristics of lig1‐fused HCVs immobilized on PS surface were compared to that of original HCV in both direct and indirect enzyme‐linked immunosorbent assay (ELISA). The results indicated that HCV‐Lig1 (Lig1 fused to HCV C‐terminus) was preferentially adsorbed on PS surface in a site‐oriented manner and would expose specific antibody‐binding sites well, which resulted in a substantial enhancement of detection sensitivity. AFM images showed that, compared to the original one, HCV‐Lig1 was arranged on PS surface in an ordered state and its conformational and steric distortions induced during the interfacial binding process were much slighter. As long as the specific epitope of a coating antigen is not located on both its N and C‐terminus, the ligand fusion approach could be an ideal strategy for site‐oriented protein immobilization. Copyright © 2010 John Wiley & Sons, Ltd.     

Separation of immunoglobulin G by high-performance pseudo-bioaffinity chromatography with immobilized histidine. I. Preliminary report on the influence of the silica support and the coupling mode.

High-performance liquid affinity chromatography with immobilized histidine as a pseudo-biospecific ligand has been used for the fractionation of human immunoglobulin G (IgG). Histidine was immobilized onto silica in two different modes: directly onto silica after epoxy activation or using an intermediate amino derivatization of silica and then coupling histidine using water-soluble carbodiimide. The behaviours and capacities of the obtained affinity supports as well as the influence of pH, silica type, pore diameter and coupling mode have been studied. IgG was effectively separated from human plasma and high maximal binding capacities were obtained.     

Covalent immobilization of glucose oxidase onto new modified acrylonitrile copolymer/silica gel hybrid supports

New polymer/silica gel hybrid supports were prepared by coating high surface area of silica gel with modified acrylonitrile copolymer. The concentrations of the modifying agent (NaOH) and the modified polymer were varied. GOD was covalently immobilized on these hybrid supports and the relative activity and the amount of bound protein were determined. The highest relative activity and sufficient amount of bound protein of the immobilized GOD were achieved in 10% NaOH and 2% solution of modified acrylonitrile copolymer. The influence of glutaraldehyde concentration and the storage time on enzyme efficiency were examined. Glutaraldehyde concentration of 0.5% is optimal for the immobilized GOD. It was shown that the covalently bound enzyme (using 0.5% glutaraldehyde) had higher relative activity than the activity of the adsorbed enzyme. Covalently immobilized GOD with 0.5% glutaraldehyde was more stable for four months in comparison with the one immobilized on pure silica gel, hybrid support with 10% glutaraldehyde and the free enzyme. The effect of the pore size on the enzyme efficiency was studied on four types of silica gel with different pore size. Silica with large pores (CPC-Silica carrier, 375 A) presented higher relative activity than those with smaller pore size (Silica gel with 4, 40 and 100 A). The amount of bound protein was also reduced with decreasing the pore size. The effect of particle size was studied and it was found out that the smaller the particle size was, the greater the activity and the amount of immobilized enzyme were. The obtained results proved that these new polymer/silica gel hybrid supports were suitable for GOD immobilization.     

Theoretical and experimental studies of biotin analogues that bind almost as tightly to streptavidin as biotin

We have used a newly developed qualitative computational approach, PROFEC (Pictorial Representation of Free Energy Changes), to visualize the areas of the ligand biotin where modifications of its structure might lead to tighter binding to the protein streptavidin. The PROFEC analysis, which includes protein flexibility and ligand solvation/desolvation, led to the suggestion that the pro-9R hydrogen atom of biotin, which is in alpha-position to the CO(2)(-) group, might be changed to a larger group and lead to better binding with streptavidin and avidin. Free energy calculations supported this suggestion and predicted that the methyl analogue should bind approximately 3 kcal/mol more tightly to streptavidin, with this difference coming exclusively from the relative desolvation free energy of the ligand. The PROFEC analysis further suggested little or no improvement for changing the pro-9S hydrogen atom to a methyl group, and great reduction in changing the ureido N-H groups to N-CH(3). Stimulated by these results, we synthesized 9R-methylbiotin and 9S-methylbiotin, and their binding free energies and enthalpies were measured for interaction with streptavidin and avidin, respectively. In contrast to the calculated results, experiments found both 9-methylbiotin isomers to bind more weakly to streptavidin than biotin. The calculated preference for the binding of the 9R- over the 9S-stereoisomer was observed. In addition, 9-methylbiotin is considerably less soluble in water than biotin, as predicted by the calculation, and the 9R isomer is, to our knowledge, thus far the tightest binding analogue of biotin to streptavidin. Subsequently, X-ray structures of the complexes between streptavidin and both 9R- and 9S-methylbiotin were determined, and the structures were consistent with those used in the free energy calculations. Thus, the reason for the discrepancy between the calculated and experimental binding free energy does not lie in unusual binding modes for the 9-methylbiotins.     

A soluble polyethyleneglycol-anchored phosphine as a highly active, reusable ligand for Pd-catalyzed couplings of aryl chlorides: comparison with cross and non-cross-linked polystyrene and silica supports

The so-called SPhos phosphine, an extremely active ligand in the amination and Suzuki coupling of sterically-hindered aryl chlorides, has been anchored on different supports such as non-soluble (cross-linked polystyrene) and soluble (non-cross-linked polystyrene and polyethyleneglycol) polymers, as well as high surface silica. SPhos anchored on polyethyleneglycol (PEG-SPhos) showed the best activity for both amination and Suzuki couplings. The PEG-SPhos ligand can be quantitatively recovered from the reaction mixture through precipitation with diethyl ether and recycled in four consecutive runs without loosing activity. ³¹P NMR spectra of the reused anchored ligand showed that deactivation of the PEG-SPhos ligand comes from the progressive oxidation of the phosphine-to-phosphine oxide.     

A spectrophotometric assay for biotinbinding sites of immobilized avidin

A rapid and sensitive spectrophotometric assay was developed for the measurement of biotin-binding sites of immobilized avidin. The method is based on the reaction of avidin with excess biotin followed by assay of the unbound biotin using the HABA (2-[4′-hydroxyazobenzene] benzoic acid) method. Three solids possessing variable amounts of monomeric avidin were examined; viz., succinamidopropyl-controlled-pore glass (CPG-500), crosslinked 6% beaded agarose (Sepharose-CL-6B**), and crosslinked bis-acrylamide/azlactone (3M Emphaze Biosupport Medium AB₁. Results indicate that the total biotin-binding sites of monomeric avidin immobilized on CPG-500, Sepharose-CL-6B, and 3M Emphaze are 0.229, 0.093, and 0.218 μmol biotin per mL beads, respectively. Assays for exchangeable biotinbinding sites gave values greater than 90% of the total sites. The spectrophotometric HABA method described is an alternative to assays based on tracers, thus the handling of radioactive material is avoided. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.     

Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography

A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively. Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylated proteins and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications of the monolith’s surface are envisaged for the future development of monoliths with improved enrichment characteristics.     

Novel and Ultrasensitive Sandwich Enzyme Immunoassay (Sandwich Transfer Enzyme Immunoassay) for Antigens

Abstract

A novel and ultrasensitive sandwich enzyme immunoassay (sandwich transfer enzyme immunoassay) for antigens is described. Antigens were reacted with dinitrophenyl monoclonal mouse antibody IgG₁ and rabbit antibody Fab′-ß-D-galactosidase conjugates. The complex formed of antigens with dinitrophenyl monoclonal mouse antibody IgG₁ and rabbit antibody Fab′-ß-D-galactosidase conjugates was trapped onto affinity-purified rabbit (antidinitrophenyl bovine serum a1bumin) IgG-coated polystyrene balls. After eliminating excess of the conjugates, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transfered to clean polystyrene balls coated with affinity-purified rabbit (anti-mouse IgG) IgG. ß-D-Galactosidase activity bound to the (anti-mouse IgG) IgG-coated polystyrene balls was assayed by fluorimetry. Nonspecifically bound ß-D-galactosidase activity considerably decreased with less decrease in specifically bound ß-D-galactosidase activity. As a result, the detection limits of human thyroid-stimulating hormone (0.01 nu, 0.02 amol) and human growth hormone (10 fg, 0.5 amol) by the present enzyme immunoassay were 30-fold lower than those by the conventional enzyme immunoassay, in which antigens were incubated with monoclonal mouse antibody IgG₁-coated polystyrene balls and rabbit antibody Fab′-ß-D-galactosidase conjugates.     

Effect of antigen size on optimal ligand density of immobilized antibodies for a high-performance liquid chromatographic support

The antigen binding capacities for purified polyclonal antibodies immobilized onto a silica-based high-performance liquid chromatographic (HPLC) affinity support are described for three serum proteins over a range of antibody ligand densities. The rate of decline in the specific activity of the immobilized antibodies with respect to increasing ligand density was found to increase with the molecular weights of the antigens. The antibodies used were purified from whole antiserum using high-performance affinity chromatography and were examined using HPLC on an SCX stationary phase. Conditions are also described for efficient coupling of the ligand to the support.     

Method to screen aromatic ligands in mixtures for quantitative affinities to target using magnetic separation of bound ligands along with HPLC and UV photometry detection

A new screening method was tested to estimate the affinity of an aromatic ligand in a mixture through competitive binding against an exogenous reference ligand. The target protein was immobilized on magnetic particles and one or several ligand(s) in each reaction mixture were prepared by parallel combinatorial-synthesis without purification. Specifically, the binding of aromatic biotin derivatives to streptavidin immobilized on magnetic particles was used as the model. An approximation equation that works under the condition of binding ratios below 0.1 for the candidate ligand and the reference ligand was developed. It was found that the relative affinity of each biotinylated aromatic candidate ligand in mixtures was consistent with that by using HPLC-MS analyses or by a homogenous method using its purified counterpart. Hence, this new screening method using HPLC-UV is considered to be highly effective.

Azide-reactive liposome for chemoselective and biocompatible liposomal surface functionalization and glyco-liposomal microarray fabrication

Chemically selective liposomal surface functionalization and liposomal microarray fabrication using azide-reactive liposomes are described. First, liposome carrying PEG-triphenylphosphine was prepared for Staudinger ligation with azide-containing biotin, which was conducted in PBS buffer (pH 7.4) at room temperature without a catalyst. Then, immobilization and microarray fabrication of the biotinylated liposome onto a streptavidin-modified glass slide via the specific streptavidin/biotin interaction were investigated by comparing with directly formed biotin-liposome, which was prepared by the conventional liposome formulation of lipid-biotin with all other lipid components. Next, the covalent microarray fabrication of liposome carrying triphenylphosphine onto an azide-modified glass slide and its further glyco-modification with azide-containing carbohydrate were demonstrated for glyco-liposomal microarray fabrication via Staudinger ligation. Fluorescence imaging confirmed the successful immobilization and protein binding of the intact immobilized liposomes and arrayed glyco-liposomes. The azide-reactive liposome provides a facile strategy for membrane-mimetic glyco-array fabrication, which may find important biological and biomedical applications such as studying carbohydrate-protein interactions and toxin and antibody screening.     

Solid-supported copper catalysts for atom-transfer radical cyclizations: assessment of support type and ligand structure on catalyst performance in the synthesis of nitrogen heterocycles

A range of solid supported pyridinemethanimine 9-11 and polyamine 12-15 ligands have been prepared on silica, polystyrene, and JandaJel supports. The CuCl and CuBr complexes of these supported ligands have been used to assess both the effect of the ligand type and the nature of the support upon a representative range of copper-mediated atom transfer 5-exo-trig 6, 24-25, 5-exo-dig 26, 4-exo-trig 28, and 5-endo-trig 27, 38 radical cyclizations to give nitrogen heterocycles. In addition, the effect of the nature of the support on the stereochemical outcome of the 5-exo cyclization of 25 has been probed. Generally, it was found that the type of support (e.g., polystyrene, silica, or JandaJel) had very little effect upon the efficiency and selectivity of the processes but that the nature of the ligand type immobilized was the important factor. Thus, the 5-exo cyclization of 6 and 24-26 proceeded more rapidly with the PMI ligands 9-11, whereas 4-exo cyclizations 28 and 5-endo radical polar crossover reactions 27 and 38 proceeded more efficiently with the JJ-TEDETA ligand 15. The efficiency of the supported ligands was also compared to their solution counterparts 4 and 5. The reusability of P-PMDETA ligand system 13 was assessed in the cyclization of 6.