Hierarchically Imprinted Polymer for Peptide Tag Recognition Based on an Oriented Surface Epitope Approach

Abstract

FLAG tag (DYKDDDDK) is a short peptide commonly used for the purification of recombinant proteins. The high price of the affinity columns and their limited reusability are a shortcoming for their widespread use in biotechnology applications. Molecularly imprinted polymers (MIPs) can circumvent some of the limitations of bioaffinity columns for such application, including long-term stability, reusability, and cost. We report herein the synthesis of MIPs selective to the FLAG tag by hierarchical imprinting. Using the epitope imprinting approach, a 5-aminoacid peptide DYKDC was selected as template and was covalently immobilized on the surface of microporous silica beads, previously functionalized with different aminosilanes namely, 3-(2aminoethylamino)propyldimethoxymethylsilane, AEAPMS, and N-(2-aminoethyl)-2,2,4-trimethyl-1-aza-2-silacyclopentane, AETAZS. We investigated the effect of the type of silane on the production of homogeneous silane grafted layers with the highest extent of silanol condensation as possible using 29 Si CP/MAS NMR. We observed that the right orientation of the imprinted cavities can substantially improve analyte recoveries from the MIP. After template and silica removal, the DYKDC-MIPs were used as sorbents for solid-phase extraction (molecularly imprinted solid-phase extraction) of the FLAG peptide, showing that the polymer prepared with AETAZS-bound silica beads contained binding sites more selective to the tag (RMIP-AZA = 87.4% vs RNIP-AZA = 4.1%, n= 3, RSD ≤ 4.2%) than those prepared using AEAPMS (RMIP-DM = 73.4% vs RNIP-DM = 23.2%, n= 3, RSD ≤ 4.0%) as functionalization agent. An extensive computational molecular modeling study was also conducted, shedding some light on the interaction mechanism between the FLAG peptide and the imprinted template in the binding cavities.

Molecular Dynamics in the Study and Development of Molecularly Imprinted Materials – Status Quo, Quo Vadis?

“…Other computational studies of MIP pre-polymerization systems have used similar methods to investigate monomer-template interactions to aid in the selection or optimization of polymer composition. The majority of these reports focused on selection of the functional monomer but examples also include choice of solvent [119,[137][138][139][140] and template [120,[140][141][142]. In some instances, similar MD simulations of systems of limited complexity were employed in order to study putative monomer-template interactions [143][144][145][146][147][148][149][150][151][152][153][154][155] or to compare different stoichiometries [156][157][158].…”

Molecularly imprinted polymers by epitope imprinting: a journey from molecular interactions to the available bioinformatics resources to scout for epitope templates

“…Template orientation control can be achieved by exploiting inhibitors, such as benzamidine, that work to directionally immobilize the template, i.e., a serineprotease enzyme via its active site [44], or more generally by coupling the epitope to a solid support in a defined direction, which is a practice that takes advantage of the classical coupling chemistries, such as carbodiimide/succinimide [25], glutaraldehyde [25], and more recently of click chemistry [17]. A further example on how to directionally immobilize an epitope lies in tagging the targeted protein with a common biochemical tag, such as the His-tag [45] or the FLAG tag [46]. Tags are convenient templates that can be immobilized by specific chemistries, such as divalent cations, and chelating affinity supports that produce pMIPs suitable to recapturing Histagged proteins [45].…”

“…Tag for protein purification -His tag epitope on the surface of the magnetic nanoparticles -His-tag-epitope imprinted magnetic nanoparticles -Specific recognition and separation of thyroid peroxidase antigens [63] as Ni 2+ , that are coordinated to an EDTA moiety immobilized on a Fe3O4@SiO2 particle [45], or the FLAG-tag [46]. A further kind of directional immobilization relies on exploiting boronic acid moieties or its derivatives, as these would provide anchoring functions to fix the sugar moieties of glycoproteins, through reversible covalent bonds.…”

The Use of Computational Methods for the Development of Molecularly Imprinted Polymers

“…MD-based investigations of other aspects of the pre-polymerization stage include studies of the structural stability of protein epitopes for template screening [ 414 , 415 ], mapping potential monomer interaction sites of a protein target, followed by docking of acrylamide-derived monomers and post-docking interaction energy calculations [ 416 ], studies of template interactions with Dengue virus as a support matrix to create larger binding sites [ 417 ], a series of reports attempting to correlate structural and physical properties of dummy templates and ligands with rebinding properties [ 418 , 419 , 420 , 421 , 422 , 423 , 424 , 425 , 426 ] and coarse-grained simulations studying the effect of composition on material properties and template interaction [ 427 ]. Additionally, large-scale MD simulations were performed in an attempt to mimic chromatography in a virtual capillary [ 428 ].…”