Reversible Immobilization Research Articles

Reversible immobilization of proteins with streptavidin affinity tags on a surface plasmon resonance biosensor chip

Dissociation of biotin from streptavidin is very difficult due to their high binding affinity. The re-use of streptavidin-modified surfaces is therefore almost impossible, making devices containing them (e.g. surface plasmon resonance (SPR) sensor chips) expensive. This paper describes a new protocol for reversible and site-directed immobilization of proteins with streptavidin affinity tags on the streptavidin-coated SPR biosensor chip (SA chip). Two streptavidin affinity tags, nano-tag and streptavidin-binding peptide (SBP tag), were applied. They both can specifically interact with streptavidin but have weaker binding force compared to the biotin-streptavidin system, thus allowing association and dissociation under controlled conditions. The SA chip surface could be regenerated repeatedly without loss of activity by injection of 50 mM NaOH solution. The fusion construct of a SBP tag and a single-chain antibody to mature bovine prion protein (scFv-Z186-SBP) interacts with the SA chip, resulting in a single-chain-antibody-modified surface. The chip showed kinetic response to the prion antigen with equilibrium dissociation constant K (D) approximately equal to 4.01 x 10(-7). All results indicated that the capture activity of the SA chip has no irreversible loss after repeated immobilization and regeneration cycles. The method should be of great benefit to various biosensors, biochips and immunoassay applications based on the streptavidin capture surface.

Isolation of polymerase chain reaction-ready bacterial DNA from Lake Baikal sediments by carboxyl-functionalised magnetic polymer microspheres

Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate- co-ethylene dimethacrylate) (P(HEMA- co-EDMA)) microspheres were used for the isolation of polymerase chain reaction (PCR)-ready DNA from samples of Baikal sediments. DNA was isolated using the phenol extraction method or the Soil DNA Isolation Kit. The occurrence of false-negative results in PCR caused by the presence of extracellular inhibitors in DNA samples was solved using solid phase reversible DNA immobilisation. PCR-ready DNA was reversibly adsorbed to the microspheres in the presence of 8.0% (w/v) poly(ethylene glycol) (PEG 6000) and 2.0 M sodium chloride concentrations. The adsorbed DNA was released from the microspheres in a low ionic strength TE buffer. The quality of isolated DNA was checked by PCR amplification.

Continuous Flow Thermal Cycler Microchip for DNA Cycle Sequencing

We report here on the use of a polymer-based continuous flow thermal cycler (CFTC) microchip for Sanger cycle sequencing using dye terminator chemistry. The CFTC chip consisted of a 20-loop spiral microfluidic channel hot-embossed into polycarbonate (PC) that had three well-defined temperature zones poised at 95, 55, and 60 degrees C for denaturation, renaturation, and DNA extension, respectively. The sequencing cocktail was hydrodynamically pumped through the microreactor channel at different linear velocities ranging from 1 to 12 mm/s. At a linear velocity of 4 mm/s resulting in a 36-s extension time, a read length of >600 bp could be obtained in a total reaction time of 14.6 min. Further increases in the flow rate resulted in a reduction in the total reaction time but also produced a decrease in the sequencing read length. The CFTC chip could be reused for subsequent sequencing runs (>30) with negligible amounts of carryover contamination or degradation in the sequencing read length. The CFTC microchip was subsequently coupled to a solid-phase reversible immobilization (SPRI) microchip made from PC for purification of the DNA sequencing ladders (i.e., removal of excess dye-labeled dideoxynucleotides, DNA template, and salts) prior to gel electrophoresis. Coupling of the CFTC chip to the SPRI microchip showed read lengths similar to that obtained from benchtop instruments but did not require manual manipulation of the cycle sequencing reactions following amplification.

Combining Enabling Techniques in Organic Synthesis: Continuous Flow Processes with Heterogenized Catalysts

The concepts article describes enabling techniques (solid-phase assisted synthesis, new reactor design, microwave irradiation and new solvents) in organic chemistry and emphasizes the combination of several of them for creating new synthetic technology platforms. Particular focus is put on the combination of immobilized catalysts as well as biocatalysts with continuous flow processes. In this context, the PASSflow continuous flow technique fulfils both chemical as well as chemical engineering requirements. It combines reactor design with optimized, monolithic solid phases as well as reversible immobilization techniques for performing small as well as large scale synthesis with heterogenized catalysts under continuous flow conditions.

Macroporous Poly(N‐isopropylacrylamide) Hydrogels with Adjustable Size “Cut‐off” for the Efficient and Reversible Immobilization of Biomacromolecules

Poly(N-isopropylacrylamide) (PNIPA) hydrogels with varied degree of crosslinking (DC) were synthesized by using poly(ethylene glycol) (PEG) as an additive. A phase separated ("macroporous") morphology was formed when using PEG contents of > or = 20 wt.-%. Temperature-dependent degrees of swelling had been measured, and average mesh sizes of the swollen polymer network had been calculated. The loading of the hydrogels with labelled dextrans with various molar masses and bovine serum albumin (BSA)-via swelling of the shrunken gel in a cold solution-and their subsequent unloading-via immersion in hot water-were studied in detail. The loading efficiencies were close to zero for PNIPA prepared at PEG contents of < or = 10 wt.-%, and they increased sharply to about 100% for PNIPA prepared with PEG contents of > or = 20 wt.-%. A complete unloading was achieved as well. For macroporous PNIPA prepared at 40 wt.-% PEG content, the loading efficiency was a function of the DC, and the "cut-off" observed as a function of dextran or protein size correlated with the mesh size of the hydrogel. The function of these "smart" hydrogels can be explained by the temperature-induced "pumping" of the solution into the gel bulk via the permanent pores, along with an uptake into the adjacent hydrogel network. Those materials could be used as matrices for the efficient and reversible immobilization of (bio)macromolecules.

Purification and preconcentration of genomic DNA from whole cell lysates using photoactivated polycarbonate (PPC) microfluidic chips

We discuss the use of a photoactivated polycarbonate (PPC) microfluidic chip for the solid-phase, reversible immobilization (SPRI) and purification of genomic DNA (gDNA) from whole cell lysates. The surface of polycarbonate was activated by UV radiation resulting in a photo-oxidation reaction, which produced a channel surface containing carboxylate groups. The gDNA was selectively captured on this photoactivated surface in an immobilization buffer, which consisted of 3% polyethylene glycol, 0.4 M NaCl and 70% ethanol. The methodology reported herein is similar to conventional SPRI in that surface-confined carboxylate groups are used for the selective immobilization of DNA; however, no magnetic beads or a magnetic field are required. As observed by UV spectroscopy, a load of ∼7.6 ± 1.6 µg/ml of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be 85 ± 5%. The immobilization and purification assay using this PPC microchip could be performed within ∼25 min as follows: (i) DNA immobilization ∼6 min, (ii) chip washout with ethanol 10 min, and (iii) drying and gDNA desorption ∼6 min. The PPC microchip could also be used for subsequent assays with no substantial loss in recovery, no observable carryover and no need for ‘reactivation’ of the PC surface with UV light.

Selective adsorption of large proteins on highly activated IMAC supports in the presence of high imidazole concentrations: Purification, reversible immobilization and stabilization of thermophilic α- and β-galactosidases

Natural proteins mainly adsorb on immobilized metal ion affinity chromatography (IMAC) supports via multipoint interactions between several His residues placed on the protein surface and different chelate moieties located on the support surface. The adsorption of large proteins on highly activated IMAC supports may involve many multi-interactions yielding a very strong adsorption. In fact, multimeric α- and β-galactosidases from Thermus sp. T2 hardly may be desorbed from highly activated IMAC supports, even in the presence of 1 M imidazole. In the presence of 50 mM of imidazole, these very large proteins can be adsorbed on these supports while the medium-small proteins hardly adsorb even on highly activated supports. In this way a very simple purification and reversible immobilization of these large proteins cloned on Escherichia coli can be performed by first heating of the crude preparation which leaves as the only large protein the thermophilic one. Interestingly, β-galactosidase from Thermus sp. T2 is 10-fold more stable than the native enzyme when incubated at 70 °C and pH 7.0. The involvement of large areas of these multimeric enzymes in the adsorption process may promote a multi-subunit adsorption with stabilizing effects. These immobilized-stabilized enzymes may be desorbed away from the support/the reactor after inactivation and a fresh solution of enzyme can be purified, immobilized and stabilized again.

Reversible Immobilization of Free-ranging African Lions (Panthera leo) with Medetomidine-tiletamine-zolazepam and Atipamezole

A combination of medetomidine-tiletamine-zolazepam was used to conduct six immobilizations of free-ranging lions (Panthera leo) in Waza National Park, Cameroon, during 1999 and 2000. Drugs were administered by dart injection at 0.07+/-0.01 (mean+/-SD) mg/kg of medetomidine and 1.8+/-0.5 mg/kg of tiletamine-zolazepam. Chemical immobilization was characterized by smooth inductions (14.1+/-6 min), satisfactory analgesia, and muscle relaxation. One animal was treated for bradypnea. No major alterations of physiologic parameters (heart and respiratory rates, rectal temperature) were seen during immobilization in the other lions. Relative arterial oxygen saturation was measured in two animals and revealed mild hypoxemia. The animals received atipamezole at 0.3+/-0.1 mg/kg intramuscularly for reversal of anesthesia. Recoveries were uneventful. All animals were radiocollared, and no mortalities occurred during an 18-mo follow-up period. Use of medetomidine-tiletamine-zolazepam for anesthesia and reversal of anesthesia with atipamezole appear to be useful for reversible immobilization of free-ranging lions.

Reversible Immobilization of Catalase by Metal Chelate Affinity Interaction on Magnetic Beads

A magnetic metal−chelate adsorbent utilizing N-methacryloyl-(l)-cysteine methyl ester (MAC) as a metal-chelating ligand was prepared. MAC was synthesized using methacryloyl chloride and l-cysteine methyl ester dihydrochloride. Magnetic beads with an average size of 150−250 μm were obtained by suspension polymerization of 2-hydroxyethyl methacrylate (HEMA) and MAC carried out in a dispersion medium. Mag-poly(HEMA−MAC) beads were characterized by surface area measurements, swelling tests, electron spin resonance (ESR) spectroscopy, elemental analysis, and scanning electron microscopy (SEM). The specific surface area of the magnetic beads was found to be 92.6 m2/g. Elemental analysis of MAC for nitrogen was estimated as 55.4 μmol/g. Then, Fe3+ ions were chelated on the magnetic beads. The Fe3+ loading was 12.7 μmol/g of support. Fe3+-chelated magnetic beads with a swelling ratio of 62% were used in the immobilization of catalase in a batch system. This approach to the preparation of enzyme carrier has several advantages over conventional immobilization methods. An expensive, time-consuming, and critical step in the preparation of immobilized metal-affinity carriers is the coupling of a chelating ligand to the adsorption matrix. In this procedure, comonomer MAC acts as the metal-chelating ligand, and it is possible to load metal ions directly on the beads without further modification steps. The maximum catalase immobilization capacity of the mag-poly(HEMA−MAC)−Fe3+ beads was observed to be 192 mg/g at pH 5.5. The Km value for immobilized catalase [mag-poly(HEMA−MAC)−Fe3+] (32.6 mM) was higher than that for free catalase (22.8 mM). Immobilized catalase exhibits enhanced stability in reaction conditions over a wide pH range (pH 5.5−7.0) and retains an activity of 76% after 10 successive batch reactions, demonstrating the usefulness of the enzyme-loaded magnetic beads in biocatalytic applications. The optimum temperature for the immobilized preparation of mag-poly(HEMA−MAC)−Fe3+−catalase at 45 °C was 5 °C higher than that of the free enzyme at 40 °C. Storage stability was found to increase with immobilization.

Purification and very strong reversible immobilization of large proteins on anionic exchangers by controlling the support and the immobilization conditions

The interaction of two large beta-galactosidases (from Escherichia coli and from Thermus sp.) with tailor-made anion exchangers was studied. Using lowly activated supports (e.g., containing 2–3 μmol of ionised groups per wet gram of support), large proteins selectively adsorbed and easily desorbed (e.g., using 200 mM of NaCl), giving highly purified proteins. However, these supports cannot be used to immobilize the enzymes for industrial use, because the weak adsorption. On the other hand, these large proteins strongly adsorb on very highly activated supports (e.g., containing 40 μmol of ionic groups per wet gram of 4 BCL agarose). Thus, these supports may be not valid for large protein purification, but may be very suitable for immobilization of these proteins. Using high ionic strength (e.g., 300 mM NaCl), large proteins still may be adsorbed on these supports, while only around 20% of total proteins adsorb, permitting some purification of the large proteins but not a total one. Moreover, adsorption under these conditions increase the adsorption strength (now there are not desorption even using 800 mM NaCl). Thus, the purification and the strong reversible immobilization of both beta-galactosidases were performed in a very simple two-step process. The large proteins can be directly adsorbed on these supports after desorption (at 200 mM of NaCl) from poorly activated supports. Furthermore, adsorption on very highly activated supports promotes a significant thermal stabilization of both enzymes, mainly in dissociations conditions.

Thermoswitched immobilization—A novel approach in reversible immobilization

The present work is based on the finding that the mesophilic carbohydrate-binding domain from Clostridium cellulovorans fused with thermophilic enzymes from Pyrococcus furiosus can be reversibly denaturated and renaturated by a simple switch of temperature. Modular recombinant enzymes are active and free in the reaction mixture at 80-90 degrees C and deactivated and immobilized by affinity adsorption on cellulose at 40-30 degrees C. The temperature transition between both modes is rather sharp and occurs within the range of 40-50 degrees C. Due to the elevated temperature, there is no limitation by a diffusion step, and contamination does not occur during the reaction. After the reaction, the enzymes are quickly deactivated, adsorbed on the affinity matrix, removed from the reaction mixture, and ready for use in another reaction cycle.

Reversible molecular immobilization on a metal surface through electrochemical reaction

The technology of mercaptide self assembly has previously been used to immobilize molecules onto epitaxial metals but is not practical for many surfaces. In this study, a designed tag was employed for the electrochemical immobilization of protein on metals (ECtag). In the case of protein, ECtag can be designed with a 6-mer amino acid (alfa-amino-1H-imidazole-4-propionic acid) homopeptide. It was employed as an ECtag ligand and was introduced to a objective protein molecule through genetical process. ECtag forms co-ordinate bonds with Ni2+ and other divalent metal ions. Protein A (specific affinity protein against igG) was chosen as a model protein for the immobilization, and was genetically tagged with an ECtag for immobilization onto a Pt electrode surface through the reduction of ECtag:Ni2+ to ECtag:Ni by the electrode potential. The immobilization process can be performed through very simple wet-electrochemical process.

Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports

In this work, we have used supports activated with m-amino-phenylboronic groups to “reversibly” immobilize proteins under very mild conditions. Most of the proteins contained in a crude extract from E. coli could be immobilized on Eupergit C-250 L activated with phenylboronic and then fully desorbed from the support by using mannitol or SDS. This suggested that the immobilization of the proteins on these supports was not only via sugars interaction, but also by other interaction/s, quite unspecific, that might be playing a key role in the immobilization of the proteins. Penicillin acylase from E. coli (PGA) was also immobilized in Eupergit C activated with m-amino-phenylboronic groups. The enzyme could be fully desorbed with mannitol immediately after being immobilized on the support. However, longer incubation times of the immobilized preparation caused a reduction of protein elution from the boronate support in presence of mannitol. Moreover, these immobilized preparations showed a higher stability in the presence of organic solvents than the soluble enzyme; the stability also improved when the incubation time was increased (to a factor of 100). By desorbing the weakest bound enzyme molecules, it was possible to correlate adsorption strength with stabilization; therefore, it seems that this effect was due to the rigidification of the enzyme via multipoint attachment on the support.

Characterization of a nucleus-encoded chitinase from the yeast Kluyveromyces lactis.

Endogenous proteins secreted from Kluyveromyces lactis were screened for their ability to bind to or to hydrolyze chitin. This analysis resulted in identification of a nucleus-encoded extracellular chitinase (KlCts1p) with a chitinolytic activity distinct from that of the plasmid-encoded killer toxin alpha-subunit. Sequence analysis of cloned KlCTS1 indicated that it encodes a 551-amino-acid chitinase having a secretion signal peptide, an amino-terminal family 18 chitinase catalytic domain, a serine-threonine-rich domain, and a carboxy-terminal type 2 chitin-binding domain. The association of purified KlCts1p with chitin is stable in the presence of high salt concentrations and pH 3 to 10 buffers; however, complete dissociation and release of fully active KlCts1p occur in 20 mM NaOH. Similarly, secreted human serum albumin harboring a carboxy-terminal fusion with the chitin-binding domain derived from KlCts1p also dissociates from chitin in 20 mM NaOH, demonstrating the domain's potential utility as an affinity tag for reversible chitin immobilization or purification of alkaliphilic or alkali-tolerant recombinant fusion proteins. Finally, haploid K. lactis cells harboring a cts1 null mutation are viable but exhibit a cell separation defect, suggesting that KlCts1p is required for normal cytokinesis, probably by facilitating the degradation of septum-localized chitin.

2,4-Dichlorophenoxyacetic acid (2,4-D) sorption and degradation dynamics in three agricultural soils

The fate and transport of 2,4-dichlorophenoxyacetic acid (2,4-D) in the subsurface is affected by a complex, time-dependent interplay between sorption and mineralization processes. 2,4-D is biodegradable in soils, while adsorption/desorption is influenced by both soil organic matter content and soil pH. In order to assess the dynamic interactions between sorption and mineralization, 2,4-D mineralization experiments were carried using three different soils (clay, loam and sand) assuming different contact times. Mineralization appeared to be the main process limiting 2,4-D availability, with each soil containing its own 2,4-D decomposers. For the clay and the loamy soils, 45 and 48% of the applied dose were mineralized after 10 days. By comparison, mineralization in the sandy soil proceeded initially much slower because of longer lag times. While 2,4-D residues immediately after application were readily available (>93% was extractable), the herbicide was present in a mostly unavailable state (<2% extractable) in all three soils after incubation for 60 days. We found that the total amount of bound residue decreased between 30 and 60 incubation days. Bioaccumulation may have led to reversible immobilization, with some residues later becoming more readily available again to extraction and/or mineralization.

Use of Medetomidine-Ketamine and Atipamezole for Reversible Immobilization of Free-ranging Hog Deer (Axis porcinus) Captured in Drive Nets

A combination of 0.05 mg/kg medetomidine and 1.5 mg/kg ketamine was used to immobilize nine adult free-ranging hog deer (Axis porcinus) captured in drive nets in the Royal Bardia National Park, Nepal, 22-23 February 2000. The drugs were administered intramuscularly from separate syringes and the mean time (+/-SD) to complete immobilization was 4.6+/-1.0 min. Muscle relaxation was good and no major clinical side effects were seen. Mean values for physiologic parameters, recorded at 10-12 and 18-20 min after drug administration, were 40.6+/-0.5 and 41.1+/-0.6 C, 87+/-5 and 84+/-4%, 107+/-16 and 113+/-16 beats/ min, and 46+/-9 and 40+/-8 breaths/min for rectal temperature, SpO2, pulse rate, and respiratory rate, respectively. All animals received 0.25 mg/ kg atipamezole intramuscularly 20-22 min after administration of medetomidine-ketamine and the mean time to coordinated running was 4.8+/-0.8 min. All animals survived for at least 5 mo post-capture. To reduce stress and to facilitate handling, medetomidine-ketamine and atipamezole are recommended for reversible immobilization of free-ranging hog deer captured in drive nets.

Magnetic hydrophilic methacrylate-based polymer microspheres for genomic DNA isolation

Carboxyl groups containing magnetic and non-magnetic microspheres were used in solid-phase reversible immobilization (SPRI) of genomic DNA. Magnetic non-porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate)--P(HEMA-co-EDMA), poly(glycidyl methacrylate)--PGMA and P(HEMA-co-GMA) microspheres with hydrophilic properties were prepared by dispersion copolymerization of the respective monomers in the presence of colloidal iron oxides. DNA from chicken erythrocytes and DNA isolated from bacterial cells of Bifidobacterium longum was used for testing of adsorption/desorption properties of magnetic microspheres. The occurrence of false negative results in polymerase chain reaction (PCR) caused by the presence of extracellular inhibitors in DNA samples has been solved using SPRI. The P(HEMA-co-EDMA) and P(HEMA-co-GMA) microspheres were used for isolation of DNA from different dairy products followed by PCR identification of Bifidobacterium strains.

Gentle cell trapping and release on a microfluidic chip by in situ alginate hydrogel formation

Microfluidic devices are increasingly used to perform biological experiments on a single-cell basis. However, long-term stability of cell positions is still an issue. A novel biocompatible method for cell entrapment and release on a microchip is presented. It is based on the controlled formation of an alginate hydrogel by bringing two laminar flows of alginate and calcium ions in the range of 2 mM to 40 mM into contact. The resulting growth of a gel bar is used to enclose and immobilize yeast cells. Adding ethylenediaminetetraacetic acid (EDTA) to the alginate solution allows for control of the hydrogel growth, and by varying the ratio of Ca(2+) to EDTA concentrations gel growth or gel shrinkage can be induced at will. Trapped cells are released during shrinkage of the gel. The trapping efficiency for different cell speeds is investigated and the properties of gel growth are discussed using a diffusion model. Precise positioning of a single cell is demonstrated. The technique presented allows not only the reversible immobilization of cells under gentle conditions but also offers the potential of long-term cell cultures as shown by on-chip incubation of yeast cells. The procedure may provide a simple and fully biocompatible technique for a multitude of innovative experiments on cells in microsystems.

Reversible and strong immobilization of proteins by ionic exchange on supports coated with sulfate-dextran.

New and strong ionic exchange resins have been prepared by the simple and rapid ionic adsorption of anionic polymers (sulfate-dextran) on porous supports activated with the opposite ionic group (DEAE/MANAE). Ionic exchange properties of such composites were strongly dependent on the size of the ionic polymers as well as on the conditions of the ionic coating of the solids with the ionic polymers (optimal conditions were 400 mg of sulfate-dextran 5000 kDa per gram of support). Around 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans could be adsorbed on these porous composites even at pH 7. This interaction was stronger than that using conventional carboxymethyl cellulose (CMC) and even others such as supports coated with aspartic-dextran polymer. By means of the sequential use of the new supports and supports coated with polyethyleneimine (PEI), all proteins from crude extracts could be immobilized. In fact, a large percentage (over 50%) could be immobilized on both supports. Finally, some industrially relevant enzymes (beta-galactosidases from Aspergillus oryzae, Kluyveromyces lactis, and Thermussp. strain T2, lipases from Candida antarctica A and B, Candida rugosa, Rhizomucor miehei, and Rhyzopus oryzae and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recoveries and immobilization rates. After enzyme inactivation, the protein could be fully desorbed from the support, and then the support could be reused for several cycles. Moreover, in some instances the enzyme stability was significantly improved, mainly in the presence of organic solvents, perhaps as a consequence of the highly hydrophilic microenvironment of the support.

Reversible immobilization of glucoamylase by ionic adsorption on sepabeads coated with polyethyleneimine.

Glucoamylase (GA) from Aspergillus niger was immobilized via ionic adsorption onto DEAE-agarose, Q1A-Sepabeads, and Sepabeads EC-EP3 supports coated with polyethyleneimine (PEI). After optimization of the immobilization conditions (pH, polymer size), it was observed that the adsorption strength was much higher in PEI-Sepabeads than in Q1A-Sepabeads or DEAE-supports, requiring very high ionic strength to remove glucoamylase from the PEI-supports (e.g., 1 M NaCl at pH 5.5). Thermal stability and optimal temperature was marginally improved by this immobilization. Recovered activity depended on the substrate used, maltose or starch, except when very low loading was used. The optimization of the loading allowed the preparation of derivatives with 750 IU/g in the hydrolysis of starch, preserving a high percentage of immobilized activity (around 50%).