Salt-induced immobilization of proteins on a high-performance liquid chromatographic epoxide affinity support

Wheatley, Jeffrey B.; Schmidt, Donald E.

doi:10.1016/0021-9673(93)80114-n

Cited by 72 publications

(48 citation statements)

References 9 publications

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“…1). These results suggest that previous physical adsorption of the enzyme is necessary to obtain covalent immobilization of proteins in epoxy supports (Scheme 1) (Katchalski-Katzir and Kraemer, 2000; Mateo et al, in press;Melander et al, 1984;Smalla et al, 1988;Wheatley and Schmidt, 1993). In fact, Figure 2 shows that, in the first step of the immobilization process, a certain percentage of the enzyme incorporated to the support could be released to the medium by incubating the enzyme derivative in the presence of imidazol.…”

Section: Selective Covalent Immobilization Of Poly His-tagged Glutarymentioning

confidence: 88%

One‐step purification, covalent immobilization, and additional stabilization of poly‐His‐tagged proteins using novel heterofunctional chelate‐epoxy supports

Mateo

Fernández‐Lorente

Cortés

et al. 2001

Biotech & Bioengineering

112

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Epoxy supports covalently immobilize proteins following a two-step mechanism; that is, the protein is physically adsorbed and then the covalent reaction takes place. This mechanism has been exploited to combine the selectivity of metal chelate affinity chromatography with the covalent immobilization capacity of epoxy supports. In this way, it has been possible to accomplish, in a simple manner, the purification, immobilization, and stabilization of a poly-His-tagged protein. To fulfill this objective we developed a new kind of multifunctional epoxy support (chelate epoxy support [CES]), which was tested using a poly-His-tagged glutaryl acylase as a model protein (an alphabeta-heterodimeric enzyme of significant industrial interest). The selectivity of the immobilization in CES toward poly-His-tagged proteins was dependent to a large extent on the density and nature of the chelated metal. The highest selectivity was achieved by using low-density chelate groups (e.g., 5 micromol/g) and metals with a low affinity (e.g., Co). However, the rate of covalent immobilization of the protein by its reaction with the epoxy groups on the support significantly increased at alkaline pH values. The multipoint attachment to the CES also depended on the reaction time. The immobilization of both glutaryl acylase subunits was achieved by incubation of the enzyme derivative at pH 10 for 24 h, with the best enzyme derivative 100-fold more stable than the soluble enzyme. By taking advantage of the selectivity properties of the novel support, we were able to immobilize up to 30 mg of protein per gram of modified Eupergit 250 using either pure enzyme or a very crude enzyme extract.

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Section: Selective Covalent Immobilization Of Poly His-tagged Glutarymentioning

confidence: 88%

One‐step purification, covalent immobilization, and additional stabilization of poly‐His‐tagged proteins using novel heterofunctional chelate‐epoxy supports

Mateo

Fernández‐Lorente

Cortés

et al. 2001

Biotech & Bioengineering

112

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“…It has, however, been reported that soluble proteins are scarcely reactive with epoxy-activated supports even under slightly alkaline conditions [15][16][17][18] . At first glance, this extremely low intermolecular reactivity between epoxy groups and nucleophiles on the protein surface constitutes a serious drawback of monofunctional epoxy supports as supports for single enzyme immobilization protocols and for more interesting enzyme immobilization-stabilization protocols.…”

Section: Immobilization Of Proteins On Monofunctional Epoxy Supportsmentioning

confidence: 99%

“…At high ionic strength, adsorption through the external hydrophobic pockets of the proteins occurs, allowing covalent attachment between nucleophiles and epoxy groups [15][16][17][18] . In fact, this kind of two-step immobilization using hydrophobic supports has already been reported for many enzymes 11,12 .…”

Section: Multipoint Covalent Immobilization Of Proteins On Heterofuncmentioning

confidence: 99%

Immobilization of enzymes on heterofunctional epoxy supports

et al. 2007

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“…It is well known epoxy groups are stable and not very reactive under mild experimental conditions. However, it has also been found that high concentrations of salt facilitate protein immobilization on epoxy-functionalized chromatographic supports [35,36] . In the hypothesized mechanism, hydrophobic interactions are enhanced in the presence of high-concentration salt.…”

Section: Protein Immobilization On Epoxysilane-modifi Ed Silica Nanopmentioning

confidence: 99%

One-step and high-density protein immobilization on epoxysilane-modified silica nanoparticles

2009

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Silica nanoparticles are most commonly modified with amino-silanes, followed by post-modification activation for protein immobilization. In this work, epoxy-functionalized silica nanoparticles were prepared by modification with glycidyloxypropyl trimethoxysilane (GPTMS) for direct protein immobilization. Silica nanoparticles possessed an average size of 46 nm, but increased to 63 nm after GPTMS modification. Reaction time, reaction temperature and GPTMS content had no significant effect on particle size. Zeta potential of SiO 2 changed from −26mV to +38mV after modification. Fourier-transformed infrared spectroscopy revealed alkyl C-H bending and stretching bands at 2944 cm −1 , 1343 cm −1 and 1465 cm −1 , respectively, for the modified nanoparticles. Fluorescein cadaverine was found to bind to GPTMS-modified SiO 2 , but not to bare SiO 2 , indicating the chemical reactivity of epoxy groups on the modified nanoparticle with amines. Finally, fluorescently labeled bovine serum albumin (BSA) was used as a model protein to investigate the capacity of epoxy-SiO 2 nanoparticles for protein immobilization. The results showed that more proteins were immobilized on the particle with longer reaction time, higher NaCl concentration, lower pH, and less GPTMS content. More importantly, proteins bound to epoxy-SiO 2 nanoparticle were highly stable. Under optimized reaction conditions, as much as 25 mg BSA/g nanoparticle was covalently attached to the nanoparticle. The epoxy silane modification of silica nanoparticles offers a reactive surface for one-step and high-density protein immobilization. epoxysilane, silica nanoparticle, protein immobilizationIn the past few years, nanomaterials of various shapes (particles, wires, rods, tubes, etc.) have been synthesized and characterized. They have been found to possess unique size-dependent properties in terms of quantum effect, specific surface area, electric conductivity, mechanical strength, and catalytic reactivity [1] . Consequently, nanomaterials have been found wide applications in the field of biological analysis and detection, biological imaging and drug delivery [2,3] . Among them, silica nanoparticles have been studied extensively due to their easy synthesis, readily available surface modification, good dispersibility and stability in water, and biocompatibility [4] . Mesoporous silica nanoparticles can be employed as drug delivery vehicles for controlled release [5] . Surface-coated silica nanoparticles act as a carrier for multiple optical [6,7] and electrochemical [8] labels to enhance detection signal. Dye-doped silica nanoparticles are more resistant to photo-bleaching than fluorescent dye molecules and much less toxic than quantum dots. They therefore offer distinct advantages when applied to in vivo fluorescence imaging [9,10] . Ruthenium tris(bipyridyl)-doped silica nanoparticles [11][12][13] have been used as electrochemiluminescent labels to provide large quantities of Ru(bpy) 3 2+ molecules for signal detection. Furthermore, silica coating on other

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Salt-induced immobilization of proteins on a high-performance liquid chromatographic epoxide affinity support

Cited by 72 publications

References 9 publications

One‐step purification, covalent immobilization, and additional stabilization of poly‐His‐tagged proteins using novel heterofunctional chelate‐epoxy supports

One‐step purification, covalent immobilization, and additional stabilization of poly‐His‐tagged proteins using novel heterofunctional chelate‐epoxy supports

Immobilization of enzymes on heterofunctional epoxy supports

One-step and high-density protein immobilization on epoxysilane-modified silica nanoparticles

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