Going native: Complete removal of protein purification affinity tags by simple modification of existing tags and proteases
| Autor: | Hui Chin Goh, Saurabh Nirantar, Farid J. Ghadessy, Radoslaw M. Sobota |
|---|---|
| Přispěvatelé: | School of Biological Sciences |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Proteases Recombinant Fusion Proteins medicine.medical_treatment Native protein Protein tag Enforced colocalisation Biology Chromatography Affinity 03 medical and health sciences Protein Domains FLAG-tag Protein purification Escherichia coli medicine Affinity tags Tandem affinity purification Protease 030102 biochemistry & molecular biology 030104 developmental biology Biochemistry Proteolysis Target protein Peptide Hydrolases Biotechnology Myc-tag |
| Zdroj: | Protein Expression and Purification. 129:18-24 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2016.09.001 |
| Popis: | Protein purification typically involves expressing a recombinant gene comprising a target protein fused to a suitable affinity tag. After purification, it is often desirable to remove the affinity tag to prevent interference with downstream functions of the target protein. This is mainly accomplished by placing a protease site between the tag and the target protein. Typically, a small oligopeptide ‘stub’ C-terminal to the cleavage site remains attached to the target protein due to the requirements of sequence-specific proteases. Furthermore, steric hindrance can also limit protease efficiency. Here, we show that respectively fusing the interacting ePDZ-b/ARVCF protein-peptide pair to the target protein and a protease enables efficient processing of a minimised sequence comprising only residues N-terminal to the cleavage site. Interaction of the protein-peptide pair enforces proximity of the protease and its minimised cleavage sequence, enhancing both catalysis of a sub-optimal site and overcoming steric hindrance. This facilitates the high yield purification of fully native target proteins without recourse to specialised purification columns. NMRC (Natl Medical Research Council, S’pore) Published version |
| Databáze: | OpenAIRE |
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