Protein purification involving a unique auto-cleavage feature of a repeated EAAAK peptide

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.