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.     

Immobilization of glucose oxidase on silica-based supports

Glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) was covalently coupled to silica-based supports containing aldehyde functional groups. The activity of the immobilized enzyme was about 1000 U/g support. The optimum pH of the catalytic activity was 5.5 for the soluble enzyme and 6.0 for the immobilized enzyme. With glucose as a substrate the Km value of the immobilized enzyme was higher than in case of the soluble enzyme. The immobilized enzyme was found to be more thermostable than the soluble one. The immobilization did not affect the stability of glucose oxidase against the denaturing effect of urea.     

Polymers containing isonitrile functional groups as supports for the covalent fixation of biologically active molecules

1) Isonitrile derivatives of synthetic polyamides, polyesters and polyalcohols, of polysaccharides such as cellulose, cross-linked dextran and agarose, linear dextran and of cross-linked and linear polyacrylamide were prepared. 2) Proteins and low-molecular-weight ligands were bound covalently to these materials by four-component reactions involving the -NC function on the polymer, amino or carboxyl groups deriving from the ligand, aldehyde and the complementary fourth component (-COOH or - NH2) being added to the aqueous reaction medium. 3) Supports of modified surface properties could be prepared by grafting of water-soluble macromolecules containing -NC groups onto preformed polymeric structures. 4) The isonitrile functional groups on polymers could be transformed into other types of functional groups by simple one-step reactions.     

Coenzymes as coribozymes

Coenzymes are small organic molecules that supply a varied set of reactive groups to protein enzymes, thereby diversifying catalysis beyond the chemistries of amino acid sidechains. As RNA structures begin with a more limited chemical diversity than proteins, it seems likely that RNA enzymes would also use functional groups from other molecules to support a complex RNA world metabolism. In fact, ribonucleotide moieties in many coenzymes have long been thought to be surviving vestiges of covalently bound coenzymes in an RNA world. The idea of coenzyme utilization by ribozymes can be explored by selection-amplification of coenzyme-binding RNAs and coenzyme-assisted ribozymes. Here, we review coenzyme-RNAs, and discuss their possible significance for RNA-mediated metabolism. In summary, a plausible route from prebiotic chemistry to ribozyme biochemistry exists for CoA, and via similar activities, likely exists for all the nucleotidyl coenzymes.     

Spin-on end-functional diblock copolymers for quantitative DNA immobilization

We demonstrate a simple means to covalently bond DNA to both hard (i.e., glass and silicon wafers) and soft (i.e., polymeric) substrates that provides quantitative and precise control of the DNA areal density. The approach is based on spin coating an alkyne-end-functional diblock copolymer, alpha-alkyne-omega-Br-poly( tBA- b-MMA), that self-assembles on both types of substrates as an ordered monolayer and thereby directs alkyne groups to the surface. Azido-functionalized DNA is covalently linked to the alkyne functionalized substrates by means of a "click" reaction between azide and alkyne groups. The density of immobilized DNA can be quantitatively controlled by varying the parameters used for spin-coating the copolymer film, that is, solution concentration and rotational speed, or by varying the copolymer molecular weight. We find the yield of the DNA coupling reaction to be dependent on the nature of the polymer underlying the reactive alkyne functional groups, being higher for more hydrophilic polymers.     

Invertase immobilization via its carbohydrate moiety

After periodate oxidation of its glycosidic component, invertase was covalently bound onto three types of modified solid supports: glycidyl methacrylate, styrene-divinylbenzene copolymers, and bead cellulose. Direct reaction of the invertase aldehyde groups that were formed with amino groups of the support and use of the modified Ugi reaction have been employed as immobilization procedures. Apart from binding methods, the important effects of the buffer, support, conditions of periodate oxidation, and the length of the spacer on the activity of the enzyme conjugate have been investigated. Superior conjugate activity was obtained, via modified Ugi reaction, by the immobilization of a suitably oxidized invertase to a styrene-divinylbenzene copolymer having free amino groups.     

Chemical probes for profiling fatty acid-associated proteins in living cells

Chemical probes appended with reactive electrophiles afford powerful tools for profiling discrete protein families in living cells. Herein, we have synthesized cell-permeable chemical probes that target fatty acid-associated proteins. These fatty acid-based chemical probes contain acyloxymethylketone or fluorophosphonate functional groups and an alkyne click chemistry tag for visualization of covalently modified proteins by in-gel fluorescence scanning. Our fatty acid-based chemical probe affords new tools to evaluate the activity/expression of lipid-associated proteins that should facilitate their functional characterization and inhibitor discovery.     

Novel histone deacetylase inhibitors: design, synthesis, enzyme inhibition, and binding mode study of SAHA-based non-hydroxamates

In order to find novel non-hydroxamate histone deacetylase (HDAC) inhibitors, a series of compounds modeled after suberoylanilide hydroxamic acid (SAHA) were designed and synthesized as (i) substrate (acetyl lysine) analogues (compounds 3–7), (ii) analogues bearing various functional groups expected to chelate zinc ion (compounds 8–15), and (iii) analogues bearing nucleophilic functional groups which could bind covalently to HDACs (compounds 16–18). In this series, semicarbazide 8b and bromoacetamides 18b,c were found to be potent HDAC inhibitors for non-hydroxamates.     

Synthesis and properties of insolubilized enzyme. XI. Trypsin-carrier-complexes on the basis of macroporous glasses by interposing the hydrophilic polymer dextran

A procedure for preparing derivatives of dialdehyde dextran-glass-carriers and their use for immobilization of trypsin is described. The dextran to the support as well as the trypsin to dextran were covalently immobilized via dialdehyde groups. The relative activity of immobilized trypsin in these enzyme-carrier-complexes amounted 17.3% and 20,4 (determined with alpha-N-Benzoyl-DL-arginine-p-nitroanilid) and 8,2% and 11.6% (determined with casein).     

Antimicrobial surfaces of viologen-quaternized poly((2-dimethyl amino)ethyl methacrylate)-Si(100) hybrids from surface-initiated atom transfer radical polymerization

To improve the antimicrobial ability of silicon-based bioelectronics and to tailor the silicon surfaces for inhibiting biofilm formation, well-defined functional polymer-Si(100) hybrids, consisting of nearly monodispersed poly((2-dimethylamino)ethyl methacrylate) (P(DMAEMA)) covalently tethered on the silicon surface and functionalized by viologen moieties, were prepared. P(DMAEMA)-Si hybrids were prepared via surface-initiated atom transfer radical polymerization (ATRP) of (2-dimethylamino)ethyl methacrylate (DMAEMA) on the hydrogen-terminated Si(100) surfaces (Si−H surfaces). The tertiary amino groups of the covalently immobilized (Si−C bonded) P(DMAEMA) brushes on the silicon substrates were quaternized by an alkyl halide to produce a high concentration of quaternary ammonium groups with biocidal functionality. Alternatively, covalent coupling of viologen moieties to the tertiary amino groups of P(DMAEMA) brushes produced the quaternized P(DMAEMA)-Si(100) hybrids with substantially enhanced antimicrobial capability, as well as capability to effectively inhibit biofilm formation. Thus, the viologen-quaternized P(DMAEMA)-Si(100) hybrids possess good antibacterial surface properties and are potentially useful to the silicon-based bioelectronics to ensure their efficiency, durability and reliability.     

Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity

Filter-paper disks of uniform size were chemically modified by the introduction of isonitrile functional groups. Avidin was then covalently linked to the disks in a four-component condensation reaction involving disk isonitrile groups and avidin carboxyl groups in the presence of a water-soluble aldehyde and an amine. Quantitative assay of unknown d-biotin solutions could be conveniently carried out with such avidin-cellulose disks by a two-step procedure: (i) immersion in the unknown sample, and (ii) exposure to an excess of radioactive biotin. Based on the known total capacity of the disks for biotin, the amount of unlabeled biotin extracted from solution by avidin-cellulose disks could be easily estimated.     

Cyanine dye labeling reagents for sulfhydryl groups

Cyanine and merocyanine dyes are introduced as new fluorescent reagents for covalently labeling proteins and other biomolecules. These dyes, which contain iodoacetamide functional groups, have high extinction coefficients and moderate quantum yields. A major advantage of these polymethine dyes is the easy manipulation of their spectral properties during synthesis. Cyanines containing reactive functional groups can be made with absorption maxima ranging from less than 500 nm to greater than 750 nm. This property opens additional regions of the spectrum for experiments involving the simultaneous multicolor analysis of different fluorescent probes. The cyanines, which are relatively insensitive to solvent property changes, are complemented by the merocyanines, which are keen indicators of solvent polarity.     

Heterofunctional supports for the one-step purification,immobilization and stabilization of large multimeric enzymes: Amino-glyoxyl versus amino-epoxy supports

For the immobilization-stabilization of multimeric enzymes, we propose a novel heterofunctional support containing a very low concentration of ionized amino groups and a very high concentration of very poorly reactive glyoxyl (aldehyde) groups. A large tetrameric enzyme, β-galactosidase from Thermus sp., was purified and dramatically stabilized with this novel support. The enzyme was first immobilized by physical adsorption via selective multipoint anionic exchange involving the largest region of the enzyme containing all enzyme subunits. Then, an additional long incubation of the immobilized derivative under alkaline conditions was performed in order to promote an intense intramolecular multipoint covalent attachment between amino groups of the adsorbed enzyme and the very stable glyoxyl groups on the support. This novel β-galactosidase derivative is the first one in which the four subunits of this enzyme become attached to a pre-existing support. Additionally, the novel amino-glyoxyl supports were much more suitable than amino-epoxy supports for intramolecular multipoint covalent immobilization of the adsorbed enzyme onto the support. In fact, at pH 7.0, the new supports covalently immobilize the physically adsorbed protein 24-fold more rapidly than epoxy supports. Furthermore, derivatives prepared on amino-glyoxyl supports preserved 85% of catalytic activity and were 5-fold more stable than derivatives prepared on amino-epoxy supports and more than 1000-fold more stable than soluble enzyme.     

Efficient synthesis of oligonucleotide conjugates on solid-support using an (aminoethoxycarbonyl)aminohexyl group for 5′-terminal modification

Solid-support conjugation at the 5′-terminal primary amine of oligonucleotides is a convenient and powerful method for introducing various functional groups. However, conventional aliphatic amines do not necessarily provide conjugates with sufficient yields. To improve the modification efficacy, we used the amino-linker (aminoethoxycarbonyl)aminohexyl group (ssH-linker), for solid-support conjugation. In the ssH-linker terminal modification, reactive free amino group could be easily presented onto a solid-support due to rapid removal of the amino-protecting group, and activated amino acids or cholesterol molecules could be covalently connected more efficiently than to typical 6-aminohexyl-linkers. Based on these results, the ssH-linker can be a useful terminal modification for the solid-support conjugation of functional molecules.     

Use of chemically modified activated carbon as a support for immobilized enzymes

Loading and activity assays of the enzymes alpha-chymotrypsin, alpha-chymotrypsinogen, and glucose oxidase covalently bound to an activated carbon support are presented. The activated carbon support material was pretreated using either a radio-frequency oxygen plasma or an electrochemical oxidation to maximize the enzyme attachment. Cyanuric chloride or water-soluble carbodiimide linking reactions were used to covalently attach the enzymes to the carbon support. Discussion of the relative merits of each reaction scheme is presented.     

New monodisperse magnetic polymer microspheres biofunctionalized for enzyme catalysis and bioaffinity separations

Magnetic macroporous PGMA and PHEMA microspheres containing carboxyl groups are synthesized by multi-step swelling and polymerization followed by precipitation of iron oxide inside the pores. The microspheres are characterized by SEM, IR spectroscopy, AAS, and zeta-potential measurements. Their functional groups enable bioactive ligands of various sizes and chemical structures to couple covalently. The applicability of these monodisperse magnetic microspheres in biospecific catalysis and bioaffinity separation is confirmed by coupling with the enzyme trypsin and huIgG. Trypsin-modified magnetic PGMA-COOH and PHEMA-COOH microspheres are investigated in terms of their enzyme activity, operational and storage stability. The presence of IgG molecules on microspheres is confirmed.     

"Glutaraldehyde-P", a stable, reactive aldehyde matrix for affinity chromatography

A high-performance affinity chromatography support based on silica has been developed for the immobilization of proteins containing primary amino groups. A hydrophilic polymer covalently bound to the silica surface minimizes nonspecific protein binding to the support while preserving high binding capacity. The Schiff base reaction involved in the coupling of a ligand to the affinity medium is rapid, allows the use of mild conditions during the coupling process, and results in a very stable linkage. Reaction parameters were studied for protein coupling to the affinity support to determine optimum binding conditions and dynamic capacity as a function of protein size. The stability of the ligand-matrix bond was determined. The performance and reproducibility of the affinity support are demonstrated by its use in the analysis of nitrophenyl sugar derivatives, purification of glycoproteins, and isolation of anti-bovine immunoglobulin G developed in rabbit.     

A novel heterofunctional epoxy-amino sepabeads for a new enzyme immobilization protocol: immobilization-stabilization of beta-galactosidase from Aspergillus oryzae

The properties of a new and commercially available amino-epoxy support (amino-epoxy-Sepabeads) have been compared to conventional epoxy supports to immobilize enzymes, using the beta-galactosidase from Aspergillus oryzae as a model enzyme. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. This support has both a great anionic exchanger strength and a high density of epoxy groups. Epoxy supports require the physical adsorption of the proteins onto the support before the covalent binding of the enzyme to the epoxy groups. Using conventional supports the immobilization rate is slow, because the adsorption is of hydrophobic nature, and immobilization must be performed using high ionic strength (over 0.5 M sodium phosphate) and a support with a fairly hydrophobic nature. Using the new support, immobilization may be performed at moderately low ionic strength, it occurs very rapidly, and it is not necessary to use a hydrophobic support. Therefore, this support should be specially recommended for immobilization of enzymes that cannot be submitted to high ionic strength. Also, both supports may be expected to yield different orientations of the proteins on the support, and that may result in some advantages in specific cases. For example, the model enzyme became almost fully inactivated when using the conventional support, while it exhibited an almost intact activity after immobilization on the new support. Furthermore, enzyme stability was significantly improved by the immobilization on this support (by more than a 12-fold factor), suggesting the promotion of some multipoint covalent attachment between the enzyme and the support (in fact the enzyme adsorbed on an equivalent cationic support without epoxy groups was even slightly less stable than the soluble enzyme).     

基于谷胱甘肽-磁性微粒的GST融合蛋白纯化体系的建立

本研究将还原型谷胱甘肽(GSH)共价结合在异硫氰酸根末端磁性微粒表面,制备了具有超顺磁性的谷胱甘肽-磁性微粒亲和介质,以表面修饰有谷胱甘肽的磁性微粒为载体,建立了谷胱甘肽巯基转移酶(GST)融合蛋白的纯化体系。对100μL细胞裂解液纯化体系所需磁性微粒用量、谷胱甘肽-磁性微粒与细胞裂解液的孵育时间、清洗条件等进行了优化。以聚丙烯酰胺凝胶电泳对融合蛋白的纯度进行了检测,Bradford方法对融合蛋白进行了定量测定,对纯化得到的目的蛋白进行了Western blotting分析。结果表明,每毫克异硫氰酸根末端磁性微粒对GSH的固定化容量为150μg,10 mg谷胱甘肽-磁性微粒可满足100μL细胞裂解体系中目的蛋白的纯化,最佳孵育时间为40 min,对GST融合蛋白的平均纯化量为516μg。本方法快速、简便,基于磁性微粒的分离还可实现自动化,对GST融合蛋白的纯化具有很好的应用前景。     

Ionic liquid-based preparation of cellulose-dendrimer films as solid supports for enzyme immobilization

Surface-active cellulose films for covalent attachment of bioactive moieties were achieved by codissolution of cellulose with polyamidoamine (PAMAM) dendrimers in an ionic liquid followed by regeneration of the composite as a film. Different generations of PAMAM were used for the formation of cellulose-dendrimer composites, as well as films with the dendrimer covalently bonded to the cellulose by means of the linker 1,3-phenylene diisocyanate. Surface characterization, thermal stability, and utility for immobilization of laccase were determined. The presence of the dendrimer amino groups was confirmed by detailed characterization of the films' surfaces. These modified films exhibit acceptable thermal stability, comparable to that of other regenerated cellulose films, but the number of active functional groups on the surface is much smaller than the theoretical amount expected. Films made with 1,3-phenylene diisocyanate as linker for covalently bound cellulose and dendrimers exhibit a better performance for immobilization of laccase than those prepared by simple mixing of the cellulose and dendrimer. In general, a linear correspondence between the dendrimer generation within the films and the specific activity of immobilized laccase in such films was not observed.