Site‐specific reversible immobilization and purification of His‐tagged protein on poly(2‐acetamidoacrylic acid) hydrogel beads

Ha, Eun-Ju; Kim, Bongsoo; Park, Eun-Kyoung; Song, Kihyung; Lee, Sun‐Gu; An, Seong Soo A.; Paik, Hyun‐jong

doi:10.1002/pat.3052

Cited by 7 publications

(4 citation statements)

References 30 publications

(47 reference statements)

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“…Recombinant his-tagged rhamnosidase was entrapped and purified using calcium alginate hydrogel beads (Puri et al 2010). Using IMAC, his-tagged green fluorescent protein and glutamyl aminopeptidase was immobilized and purified within nickel functionalized poly(2-acetamidoacrylic acid) hydrogels and hydrogel beads (Ha et al 2008(Ha et al , 2012(Ha et al , 2013. Poly(ethylene glycol)-based hydrogels modified with metalaffinity ligands were employed for binding and release of histagged GFP Metters 2007, 2008).…”

Section: His-tag Adsorption and Protonationmentioning

confidence: 99%

Adsorption and protonation of peptides and proteins in pH responsive gels

Longo¹,

Szleifer²

2016

J. Phys. D: Appl. Phys.

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To describe the non-trivial features of the equilibrium protonation and physical adsorption of peptides/proteins in pH-responsive hydrogels, we summarize our recent theoretical work on the subject. In these systems, molecular confinement in nanometer-sized environments modifies the balance between chemical state, physical interactions and molecular organization, which results in a behavior that is qualitatively different from what is expected from assuming the bulk solution protonation. To enhance adsorption, the pH-dependent deprotonation curves of all amino acids of adsorbed proteins are adequately shifted and deformed, which depends, in a complex fashion, on the specific amino acid. This possibility of modifying different acid-base equilibriums gives the adsorbed protein degrees of freedom to regulate charge and enhance electrostatic attractions under a wide range of experimental conditions. Protein adsorption modifies the microenvironment inside the hydrogel, particularly the gel pH. As a result, the state of protonation of the network is different before and after adsorption. The physicochemical considerations described in this review can be useful in the design of functional materials involving protein adsorption.

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Section: His-tag Adsorption and Protonationmentioning

confidence: 99%

Adsorption and protonation of peptides and proteins in pH responsive gels

Longo¹,

Szleifer²

2016

J. Phys. D: Appl. Phys.

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“…One-step immobilization and purification methods have been applied before for a Histagged transaminase [41], a histidine-tagged green fluorescent protein [72], an extracellular His-tagged Thermomyces lanuginosus lipase [73], and even for the co-immobilization of two components of hydroxylase monooxygenase that were employed to construct a bienzymatic catalytic system [46]. Moreover, Zhou et al studied the selective purification and immobilization of a His-tagged recombinant glucose dehydrogenase onto NiFe 2 O 4 magnetic nanoparticles in a one-step procedure [74].…”

Section: Immobilization Of Recombinant Gentisate 12-dioxygenasementioning

confidence: 99%

Characterization of Gentisate 1,2-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and Its Stabilization by Immobilization on Nickel-Functionalized Magnetic Nanoparticles

Asimakoula

Giannakopoulou

Lappa

et al. 2022

Applied Microbiology

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The aim of this study was the biochemical and kinetic characterization of the gentisate 1,2-dioxygenase (GDO) from Pseudarthrobacter phenanthrenivorans Sphe3 and the development of a nanobiocatalyst by its immobilization on Ni2+-functionalized Fe3O4-polydopamine magnetic nanoparticles (Ni2+-PDA-MNPs). This is the first GDO to be immobilized. The gene encoding the GDO was cloned with an N-terminal His-tag and overexpressed in E. coli. The nanoparticles showed a high purification efficiency of GDO from crude cell lysates with a maximum activity recovery of 97%. The immobilized enzyme was characterized by Fourier transform infrared spectroscopy (FTIR). The reaction product was identified by 1H NMR. Both free and immobilized GDO exhibited Michaelis–Menten kinetics with Km values of 25.9 ± 4.4 and 82.5 ± 14.2 μM and Vmax values of 1.2 ± 0.1 and 0.03 ± 0.002 mM*s−1, respectively. The thermal stability of the immobilized GDO was enhanced at 30 °C, 40 °C, and 50 °C, compared to the free GDO. Stored at −20 °C, immobilized GDO retained more than 60% of its initial activity after 30 d, while the free enzyme completely lost its activity after 10 d. Furthermore, the immobilized nanoparticle–enzyme conjugate retained more than 50% enzyme activity up to the fifth cycle.

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“…46 As stated in the previous paragraph, pH-sensitive hydrogels have been recently applied to design full three-dimensional environments suitable for the manipulation of proteins. [41][42][43]46 Although the physical properties of these materials can be externally tuned by simply changing the composition of the solvent, this responsive behavior has not been exploited in the separation/immobilization of proteins. The long-term motivation for this work is to investigate the possibility of using the solution to enhance or control the adsorption of biomolecules into pH-responsive hydrogels.…”

Section: ■ Introductionmentioning

confidence: 99%

“…The host environment can help enzymes retain their activity, stabilizing them against thermally induced denaturation and aggregation, as well as promote refolding of acid-denatured proteins. , Therefore, hydrogels are excellent candidates for the development of new materials that can serve as the biocompatible matrix for the chromatography of biomolecules. Immobilization and purification of his 6 -tagged proteins and enzymes within nickel functionalized poly(2-acetamidoacrylic acid) hydrogels have been demonstrated. − Poly(ethylene glycol) hydrogels, whose polymer network has been modified with ligands for metal affinity, have been used for the controlled binding and release of hexahistidine-tagged proteins. , Moreover, using the high affinity between histidine residues and a metal-ion complex, his-tagged rhamnosidase was immobilized within calcium alginate hydrogel beads …”

Section: Introductionmentioning

confidence: 99%

Equilibrium Adsorption of Hexahistidine on pH-Responsive Hydrogel Nanofilms

Longo

Cruz²,

Szleifer³

2014

Langmuir

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We present a molecular theory to study the adsorption of different species within pH-sensitive hydrogel nanofilms. The theoretical framework allows for a molecular-level description of all the components of the system, and it explicitly accounts for the acid-base equilibrium. We concentrate on the adsorption of hexahistidine, one of the most widely used tags in bio-related systems, particularly in chromatography of proteins. The adsorption of hexahistidine within a grafted polyacid hydrogel film shows a nonmonotonic dependence on the solution pH. Depending on the salt concentration, the density of the polymer network, and the bulk concentration of peptide, substantial adsorption is predicted in the intermediate pH range where both the network and the amino acids are charged. To enhance the electrostatic attractions, the acid-base equilibrium of adsorbed hexahistidine is shifted significantly, increasing the degree of charge of the residues as compared to the bulk solution. Such a shift depends critically on the conditions of the environment at the nanoscale. At the same time, the degree of dissociation of the network becomes that of the isolated acid group in a dilute solution, which means that the network is considerably more charged than when there is no adsorbate molecules. This work provides fundamental information on the physical chemistry behind the adsorption behavior and the response of the hydrogel film. This information can be useful in designing new materials for the purification or separation/immobilization of histidine-tagged proteins.

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Site‐specific reversible immobilization and purification of His‐tagged protein on poly(2‐acetamidoacrylic acid) hydrogel beads

Cited by 7 publications

References 30 publications

Adsorption and protonation of peptides and proteins in pH responsive gels

Adsorption and protonation of peptides and proteins in pH responsive gels

Characterization of Gentisate 1,2-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and Its Stabilization by Immobilization on Nickel-Functionalized Magnetic Nanoparticles

Equilibrium Adsorption of Hexahistidine on pH-Responsive Hydrogel Nanofilms

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