Optimal spacer arm microenvironment for the immobilization of recombinant Protein A on heterofunctional amino-epoxy agarose supports
Introduction
One of the main applications of protein-ligand immobilization is the affinity chromatography for antibody purification [1,2]. The development of protocols improving the binding capacity and decreasing the ligand leakage is therefore a very exciting goal in pharmaceutical sciences and biotechnology [3,4]. Theoretically, oriented immobilization of proteins on supports, exposing the activity center to the medium, is conducive to making full use of the binding activity of ligands [[5], [6], [7]]. Moreover, the protein immobilization through many covalent linkages could prevent the ligand leaking from the adsorbent [8,9]. Therefore, an optimal immobilization strategy for preparing the affinity adsorbent is supposed to involve two aspects: multipoint covalent immobilization and site-specific immobilization where the reactive residues are far from the activity center of proteins. Heterofunctional support, bearing the physically adsorptive groups and chemical reactive groups, is one promising candidate to fulfill the requirements [[10], [11], [12], [13]]. Proteins could be covalently immobilized on the heterofunctional support through the “two-step immobilization” mechanism [10,14,15]: firstly, proteins are quickly adsorbed on the support by the physically adsorptive groups of the support; secondly, the protein surface region near the support is covalently attached to achieve the oriented immobilization. Additionally, the multipoint covalent immobilization occurs while carrying out the long-term incubation of proteins and heterofunctional supports.
Depending on the nature of proteins and the experiment objective, diverse heterofunctional supports bearing different adsorptive groups and reactive groups have been developed [16,17]. Tailor-made heterofunctional activated octyl-supports with the reactive groups of glyoxyl, divylnylsulfone or epoxy, were used to immobilize the protein with the hydrophobic surface region [[18], [19], [20], [21]]. For the hydrophilic proteins with the isoelectric point less than 7, heterofunctional supports combining the amino groups and various reactive groups were applied. Additionally, due to the reversible covalent interaction of acyl groups and proteins [20,22], heterofunctional supports bearing acyl chains and other reactive groups, such as acyl-glyoxyl, acyl-epoxyde, acyl-vinylsulfone or acyl-glutaraldehyde, have been recently proposed [[23], [24], [25], [26], [27]]. Of these heterofunctional supports, amino-epoxy supports seem to be almost ideal matrices to prepare the protein ligand based affinity adsorbent [14,[28], [29], [30]]. Epoxy groups may react with many nucleophilic groups present on the protein surface to form extremely strong covalent attachment [14,31,32]. In addition, epoxy groups are very stable, making it possible to perform long-term incubations of immobilized proteins in order to get an intense multipoint covalent attachment [33,34]. Although the intermolecular reactivity between epoxy supports and proteins is very low [10], the multipoint covalent immobilization of proteins on the heterofunctional amino-epoxy support could be promoted by the “two-step immobilization” mechanism [35]. Following the first immobilization of ionic adsorption by the amino group, intramolecular reactivity between the epoxy groups of the support and the nucleophiles of adsorbed proteins placed in the vicinity of the supports occurs [30]. Thus, it may be expected that the oriented immobilization of proteins on the supports could be realized by the heterofunctional amino-epoxy support.
The conventional heterofunctional amino-epoxy support was prepared by the modification of some epoxy groups with ethylenediamine [28,36]. In order to preserve the maximum amount of epoxy groups to achieve the covalent attachment between the protein and the support, a very small number of the amino groups are present on the conventional heterofunctional amino-epoxy support [14]. In addition, novel heterofunctional supports of amino-epoxy Sepabeads [37] and MANAE-epoxy agarose [36,38] were reported to be used for immobilizing enzymes. These heterofunctional amino-epoxy supports exhibit different properties of enzymes immobilization as the amino and epoxy groups are in different microenvironment. However, the effects of the spacer arm microenvironment of different heterofunctional amino-epoxy supports on the proteins immobilization have not been investigated in detail.
The heterofunctional support was widely used for enzyme immobilization and has a plethora of applications in biocatalysis, biosensor development, decontamination and energy storage [[39], [40], [41], [42], [43], [44]]. However, these supports are rarely used for the affinity chromatography. Especially, in the case of antigen-antibody based affinity chromatography, as the protein ligand to be immobilized needs to recognize very large substrates, the orientation of the immobilized protein regarding the support is a key point: only if the activity center is exposed to the medium, the protein will be able to bind these large substrates [11]. The protein immobilization strategy significantly affects the adsorption properties of affinity materials. Therefore, investigations into the effects of the spacer arm microenvironment of heterofunctional supports on the protein immobilization and consequent binding capacity of affinity materials are essential to develop the affinity chromatography.
Staphylococcal Protein A (SPA) affinity chromatography is a well-established platform for purification of clinical-grade antibodies and Fc-containing proteins due to its highly specific nature [45,46]. Native SPA, a cell surface protein expressed by Staphylococcus aureus, comprises a cell wall binding domain designated X and five highly homologous Fc binding domains designated E, D, A, B, and C [47]. The five Fc binding domains are organized in an anti-parallel three-helical arrangement each, showing individual affinity for the Fc-fragment residues of IgG from most mammalian species [48,49]. For the purpose of improving the binding capacity and stability, engineering recombinant SPA (rSPA), usually a tandem of B domain or Z(N23 T, F30A) domain (a mutant domain derived from B-domain) [50,51], has been applied to the ligand of affinity adsorbent instead of native SPA. In addition, structures of supports are very essential for the properties of the affinity chromatography [44,52]. Sepharose, a kind of agarose bead, has been widely used as the support of affinity adsorbents due to the good hydrophilic, inert and mechanical properties, as well as the abundant reactive hydroxyl groups and proper pore sizes [53].
In this work, we prepared three heterofunctional amino-epoxy agarose supports: traditional amino-epoxy (AE) agarose prepared by the partially amination of epoxy agarose, glycidyl-amino (GA) agarose by the epichlorohydrin (ECH) modification of amino agarose, and butanediol diglycidyl-amino (BDA) agarose by the 1,4-butanediol diglycidyl ether (BD) modification of amino agarose. Compared with the traditional AE agarose, the BDA and GA agaroses bear the epoxy and amino group in the same spacer arm. The different spacer arm microenvironments are expected to exhibit different immobilization behaviors for proteins. Specifically, rSPA, a tandem of three B domains [54], was immobilized on the three heterofunctional supports to prepare affinity adsorbents. Influences of the spacer arm microenvironment on the rSPA immobilization were discussed. Adsorption performances of human IgG were studied carefully to evaluate the heterofunctional amino-epoxy agarose suitable for the rSPA affinity chromatography.
Section snippets
Materials and reagents
Agarose support (Sepharose 6FF, 45–165 μm) was purchased from GE Healthcare (Sweden). Fresh-frozen human plasma was obtained from a local plasma donation center. Human IgG (hIgG) used in this work was purified from human plasma, with the purity of ≥ 95 % by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An extinction coefficient at 280 nm of 1.36 (mg/mL)−1 cm−1 was used for the hIgG [49]. The rSPA was purified by the metal affinity chromatography as previously described [
Preparation of heterofunctional amino-epoxy agaroses
Three heterofunctional agarose supports with an approximate 1:1 molar ratio of amino groups to epoxy groups were prepared. In the case of AE agarose, in order to have the enough epoxy groups, the agarose was activated with ECH using DMSO as solvent and obtained the epoxy agarose with the epoxy density of 68 μmol/mL. By controlling the amination time, half of epoxy groups were aminated with ethylenediamine to obtain the AE agarose with approximately equal density of 34 μmol/mL for the amino and
Conclusions
In summary, three heterofunctional amino-epoxy agaroses with the different spacer arm microenvironment were prepared for rSPA affinity adsorbents. Hydrophobic butylidene groups on the BDA agarose have a negative effect on the first immobilization of rSPA, which leads to the low immobilization yield of rSPA on the support. Compared with the GA agarose, the ionized amino group on the AE agarose (a traditional heterofunctional support) is closer to the solid support, which slightly impedes the
Declaration of Competing Interest
The authors declare that they have no conflict of interest.
Acknowledgements
This work was financially supported by National Natural Science Foundation of China (Grant Nos. 21965039 and 21464017), Natural Science Foundation of Yunnan Province (Grant Nos. 2014FB139 and 2018FD017), and Science Research Foundation of Yunnan Education Bureau (Grant No. 2014Z046).
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2020, International Journal of Biological MacromoleculesCitation Excerpt :Furthermore, it is possible to change the orientation of enzymes through immobilization conditions (especially ionic strength), favoring one mechanism or another as the primary cause of immobilization. The disadvantage is that the different forms of immobilization associated with the support allow that the enzymes to have different orientations, exhibiting different stability and catalytic behavior [17,18]. The choice of solid heterofunctional support that acts as a skin dressing under the wound can be advantageous in the healing process, especially during the cleaning of the injured area [15–17].