標題: Protein purification involving a unique auto-cleavage feature of a repeated EAAAK peptide
作者: Wu, Yue-Jin
Fan, Chia-Ying
Li, Yaw-Kuen
應用化學系
Department of Applied Chemistry
關鍵字: Protein purification;Peptide linker;Auto-cleavage;Chitin column
公開日期: 1-十二月-2009
摘要: 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. (C) 2009 Elsevier B.V. All rights reserved.
URI: http://dx.doi.org/10.1016/j.jchromb.2009.10.009
http://hdl.handle.net/11536/6327
ISSN: 1570-0232
DOI: 10.1016/j.jchromb.2009.10.009
期刊: JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume: 877
Issue: 31
起始頁: 4015
結束頁: 4021
顯示於類別:期刊論文


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