Elsevier

Journal of Chromatography A

Volume 1592, 10 May 2019, Pages 192-196
Journal of Chromatography A

Site-specific immobilization of lysozyme upon affinity chromatography resin by forecasting lysine activity and controlling pH and epoxy group density

https://doi.org/10.1016/j.chroma.2019.02.066Get rights and content

Highlights

  • A site-specific immobilization strategy for protein chromatographic ligand.

  • Forecasting activity order of immobilization sites by molecular simulation.

  • Major influence factor for acitivity of immobilization sites is pH micro-environment.

  • Quantitatively detecting of immobilization sites by comparing the peptide maps.

  • Site-specific immobilization of lysozyme by controlling pH and epoxy group density.

Abstract

Here, a site-specific immobilization strategy for lysozyme was discussed. First, we calculated the surface micro-environment of lysozyme as a model protein and forecasted the nucleophilic attack activity order of six lysine residues within lysozyme, namely, K96> K97 and K33>K1, K13, and K116. Second, lysozyme was immobilized on agarose resin (epoxy group density 22.34 μmol/g) and incubated at pH 9.5 for 12 h. We then compared the peptide maps of free and immobilized lysozyme and established a quantitative detection method for immobilization sites. Third, we studied the effect of immobilization conditions such as epoxy group density and pH upon immobilization sites. When lysozyme was immobilized upon resin with a high epoxy group density (22.34 μmol/g) at pH 9.5 for 12 h, multiple lysozyme immobilization sites were found, including K33, K96 and K97; when lysozyme was immobilized upon resin with a low epoxy group density (11.36 μmol/g) at pH 9.5 for 12 h, only one lysozyme immobilization site was found, K96. The pH also had an effect upon immobilization sites. When immobilization was performed at pH≥10.5 with low epoxy group density resin (11.36 μmol/g) for 12 h, the immobilization sites included at least K33, K96 and K97; when immobilization was performed at pH 9.5 with other conditions remaining unchanged, the only lysozyme immobilization site found was at K96. These experimental results were highly consistent with the forecasted results, revealing some regularities of immobilization site control for lysozyme upon affinity chromatography resin.

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)).

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