Protein purification involving a unique auto-cleavage feature of a repeated EAAAK peptide
| Autor: | Yue-Jin Wu, Yaw-Kuen Li, Chia Ying Fan |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Recombinant Fusion Proteins Clinical Biochemistry Chitin Bioengineering Peptide Applied Microbiology and Biotechnology Biochemistry Analytical Chemistry Column chromatography Bacterial Proteins Protein purification Cloning Molecular Tandem affinity purification chemistry.chemical_classification Expression vector Chromatography Chemistry Cell Biology General Medicine Hydrogen-Ion Concentration Fusion protein Target protein Peptides Linker Biotechnology |
| Zdroj: | Journal of Chromatography B. 877:4015-4021 |
| ISSN: | 1570-0232 |
| DOI: | 10.1016/j.jchromb.2009.10.009 |
| Popis: | Protein purification generally requires many steps of column chromatography that typically involve ion-exchange, hydrophobic-interaction and gel-filtration separations. More sophisticated purification of protein might be achieved through an application of affinity binding on a functionalized gel such as a nickel column, glutathione-modified column, maltose-modified gel column or others. Of several drawbacks existing in these methods, fusion proteins are commonly obtained, protease digestion might be necessary to remove the fusion moiety; a costly gel is employed for affinity binding, etc. Here we report that an expression vector derived from pREST was constructed to compose the gene of the chitin-binding protein (CBP) and the nucleotide sequence of the (EAAAK)(5) peptide linker following restriction sites for target gene insertion. Fusion proteins were expressed with E. coli and purified with a chitin column. The (EAAAK)(5) linker is shown to possess a pH-dependent auto-cleavage feature. In the range pH 6-7, the target protein becomes automatically released from the fusion protein without proteolytic treatment. Although the mechanism of this auto-cleavage property of an (EAAAK)(5) linker is unclear, this feature has been successfully employed for many cases of protein purification without the tag of a fusion protein. |
| Databáze: | OpenAIRE |
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