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.
Department of Applied Chemistry, National Chiao Tung University, 1001 Ta-Hseh Rd., Hsinchu 30050, Taiwan.
Yue-Jin
YJ
Wu
Chia-Ying
CY
Fan
Yaw-Kuen
YK
Li
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002686:Chitin / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D001426:Bacterial Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D002686:Chitin