Site-specific immobilization of lysozyme upon affinity chromatography resin by forecasting lysine activity and controlling pH and epoxy group density
Introduction
Protein immobilization is an important technique in the biomaterial field and has been widely used for industrial applications such as affinity chromatography resin, immobilized enzymes, biosensors and bioreactors [1]. In the immobilization process, it was very important to control the immobilization sites of proteins and obtain homogeneously oriented proteins, which could significantly improve the activities and efficiencies of protein-immobilized devices [2,3].
However, site-specific immobilization still faces some challenges. In the original strategy of chemical immobilization, lysine, owing to its epsilon-amino group, was the most important optional immobilization site. Unfortunately, there is always more than one lysine residue in any protein molecule, and the traditional method could provide neither predictability nor good selectivity for immobilization sites, making site-specific control very difficult. Recently, some researchers have developed novel strategies for site-specific immobilization, but the working areas were generally limited. Yang [4] employed a Z-domain to assist site-specific 3D immobilization for IgG and obtained satisfactory results. However, this strategy was not applicable to other proteins. Zang [5], Raeeszadeh-Sarmazdeh [6], Leidner [7], and Li [8] successfully achieved site-specific immobilizations of several proteins with assistance from different fusion tags. Lee [3] designed a mutant of bovine carbonic anhydrase II with a single mutation to Cys and achieved site-specific immobilization. Obviously, these methods were not applicable to natural proteins.
On the other hand, accurate analysis of protein immobilization sites was not mature enough, largely challenging the research controlling site specificity. 2D electrophoresis [9], NMR [10] and surface-enhanced Raman scattering spectroscopy [11] were employed to monitor the orientation of protein immobilized upon substrate. Unfortunately, most of these methods could not identify the immobilizing amino acid sites and had some limitations in quantitative analysis.
To address these challenges, we used immobilization of lysozyme upon agarose chromatography resin as a model to study a universal strategy for controlling the immobilization sites, including a novel method evaluating the activity order of lysine residues and a quantitative method monitoring the immobilization sites.
Section snippets
Materials
Lysozyme with 93% purity was purchased from Amresco (USA). Trypsin and chymotrypsin were purchased from Promega (USA). Agarose 4 F F chromatographic resin was provided by the National Engineering Research Center for Biotechnology, Beijing, China. Dithiothreitol (DTT), 1,4-butanediol diglycidyl ether (BDGE), trifluoroacetic acid (TFA), and acetonitrile (ACN) were purchased from Thermo Fisher (USA). All other chemicals were analytical grade reagents. All solutions were made using Milli-Q grade
Micro-environment simulation and activity forecasting of lysine residues
In this work, lysozyme could be immobilized upon epoxy-activated resin through nucleophilic attack by ε-amino groups in six candidate lysine residues, whose attack activities were uneven. Here, the order of nucleophilic attack activities was analysed using molecular simulation. The activity difference should be related to lysine residue situations and surface micro-environments, as free lysine residues exhibited identical pKa values. We calculated the activities of six lysine residues using
Conclusion
It was practical to forecast the activities of lysine residues in protein molecules using molecular simulation. The activity order was K96 > K97 and K33 > K1, K13, and K116 in lysozyme. It was also practical to quantitatively analyse the immobilization sites within the protein molecule upon affinity chromatography resin using HPLC-MS and restriction enzymes. The epoxy group densities had an effect upon immobilization sites. When the epoxy group density was 11.36 μmol/g, lysozyme molecules were
Acknowledgements
This research was financially supported by the National Key Technology R&D Programs of China (2018YFA0108203 and 2018YFC1106402) and Guangzhou People's Livelihood Science and Technology Project of China (201803010086)).
References (14)
- et al.
Supramolecular strategies for protein immobilization and modification
Curr. Opin. Chem. Biol.
(2018) - et al.
Site-directed antibody immobilization techniques for immunosensors
Biosens. Bioelectron.
(2013) - et al.
Stabilization of Bovine carbonic anhydrase II through rational site-specific immobilization
Biochem. Eng. J.
(2018) - et al.
Site-specific covalent attachment of an engineered Z-domain onto a solid matrix: an efficient platform for 3D IgG immobilization
Anal. Chim. Acta
(2015) - et al.
Direct site-specific immobilization of protein A via aldehyde-hydrazide conjugation
J. Chromatogr. B
(2016) - et al.
Site-specific immobilization of protein layers on gold surfaces via orthogonal sortases
Colloids Surf. B Biointerfaces
(2015) - et al.
Oriented immobilization of a delicate glucose-sensing protein on silica nanoparticles
Biomaterials
(2019)
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2022, Colloids and Surfaces B: BiointerfacesCitation Excerpt :Multipoint immobilization is conducive to improving immobilization efficiency. According to Zhang et al. [40], when the epoxy group density on the surface of the agarose support is 22.34 μmol g−1, three lysozymes can be immobilized at the same time, but when the epoxy density is 11.36 μmol g−1, only one lysine of lysozyme participates in the immobilization. However, the efficiency of mass-transfer of substrates to the immobilized enzymes may be affected due to the rigidity of immobilized enzymes, as the small swing angle of multi-point immobilized enzymes lead to less chance of contact between active centers and substrates [37].
Oriented immobilization of antibodies through different surface regions containing amino groups: Selective immobilization through the bottom of the Fc region
2021, International Journal of Biological MacromoleculesCitation Excerpt :The immobilization rate of anti-HRP (pI = 5.2–6.55) using different methods was evaluated (Fig. 3). Antibodies can be immobilized on the monofunctional support directly, and the immobilization rate depends significantly on the incubation condition used and the density of functional groups located on the support and proteins [40,41]. When incubated under lower alkaline buffer (pH 8.5), extreme N-termini are preferentially attached to glyoxyl-activated resin because of the low pKa of the α-amino group [27].
Optimal spacer arm microenvironment for the immobilization of recombinant Protein A on heterofunctional amino-epoxy agarose supports
2020, Process BiochemistryCitation Excerpt :Compared with the reported amino-epoxy agarose supports, the AE, GA and BDA agarose supports present equal and high densities of both amino and epoxy groups, benefitting from the high density epoxy agarose prepared by the agarose activated with ECH using DMSO as the solvent [8]. The immobilization of proteins on the amino-epoxy agarose correlates with the ion strength and pH of the solution [5]. The low ion strength and pH between the isoelectric point (pI) of proteins and the pK of amino groups on supports are adopted for the immobilization.