Tag-specific affinity purification of recombinant proteins by using molecularly imprinted polymers
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 polyhistidine tag. However, purification requires...
| Autores: | , , , |
|---|---|
| Tipo de documento: | artigo |
| Data de publicação: | 2019 |
| País: | España |
| Recursos: | Universidad Complutense de Madrid (UCM) |
| Repositório: | Docta Complutense |
| Idioma: | inglês |
| OAI Identifier: | oai:docta.ucm.es:20.500.14352/93327 |
| Acesso em linha: | https://hdl.handle.net/20.500.14352/93327 |
| Access Level: | Acceso aberto |
| Palavra-chave: | 543 Molecularly imprinted polymers Epitope imprinting FLAG tag Synthetic receptors Protein purification Química analítica (Química) 23 Química 24 Ciencias de la Vida 33 Ciencias Tecnológicas 32 Ciencias Médicas |
| Resumo: | 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 polyhistidine tag. However, purification requires using anti-FLAG antibody resins, the high cost and non-reusability 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. |
|---|