Epitope tagging is widely used to fuse a known epitope to proteins for which no affinity receptor is available by using recombinant DNA technology. One example is FLAG epitope (DYKDDDDK), which provides better purity and recoveries than the favorite poly histidine tag. However, purification requires using anti-FLAG antibody resins, the high cost and nonreusability of which restrict widespread use. One cost-effective solution is provided by the use of bioinspired anti-FLAG molecularly imprinted polymers (MIPs). This work describes the development of MIPs, based on the epitope approach, synthesized from the tetrapeptide DYKD as template that affords purification of FLAG-derived recombinant proteins. Polymer was optimized by using a combinatorial approach to select the functional monomer(s) and cross-linker(s), resulting in the best specific affinity toward FLAG and the peptide DYKD. The imprinted resin obtained was used to purify mCherry proteins tagged with either FLAG or DYKD epitopes from crude cell lysates. Both mCherry variants were highly efficiently purified (R ≥ 95%, RSD ≤ 15%, n = 3) and impurities were removed. Unlike existing antibody-based resins, the proposed tag-imprinting strategy provides a general method for meeting the growing demand for efficient, inexpensive, and versatile materials for tagged proteins purification.

No abstract

Food allergy is one of the major health threats for sensitized individuals all over the world and, over the years, the food industry has made significant efforts and investments to offer safe foods for allergic consumers. The analysis of the concentration of food allergen residues in processing equipment, in raw materials or in the final product, provides analytical information that can be used for risk assessment as well as to ensure that food-allergic consumers get accurate and useful information to make their food choices and purchasing decisions. The development of biosensors based on nanomaterials for applications in food analysis is a challenging area of growing interest in the last years. Research in this field requires the combined efforts of experts in very different areas including food chemistry, biotechnology or materials science. However, the outcome of such collaboration can be of significant impact on the food industry as well as for consumer’s safety. These nanobiosensing devices allow the rapid, selective, sensitive, cost-effective and, in some cases, in-field, online and real-time detection of a wide range of compounds, even in complex matrices. Moreover, they can also enable the design of novel allergen detection strategies. Herein we review the main advances in the use of nanoparticles for the development of biosensors and bioassays for allergen detection, in food samples, over the past few years. Research in this area is still in its infancy in comparison, for instance, to the application of nanobiosensors for clinical analysis. However, it will be of interest for the development of new technologies that reduce the gap between laboratory research and industrial applications.

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.

No abstract