Journal of Biotechnology 2011-03-10

Site-specific tetrameric streptavidin-protein conjugation using sortase A.

Takuya Matsumoto, Shiori Sawamoto, Takayuki Sakamoto, Tsutomu Tanaka, Hideki Fukuda, Akihiko Kondo

文献索引：J. Biotechnol. 152 , 37-42, (2011)

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摘要

Streptavidin is tetrameric protein which has tight and specific biotin binding affinity, and streptavidin modification of proteins or small molecules is widely used for biotechnology tool. Here, we demonstrate site-specific streptavidin-protein conjugation using enzymes. We focused on sortase A, a transpeptidase from Staphylococcus aureus. A streptavidin-tagged LPETG motif (Stav-LPETG) was expressed in Escherichia coli. We achieved soluble streptavidin expression in E. coli without refolding using a cold shock expression system. Then we successfully conjugated Stav-LPETG with pentaglycine-appended green fluorescence protein (Gly5-GFP) or triglycine-appended glucose oxidase (Gly3-GOD) using sortase A. SDS-PAGE analysis showed site-specific tetrameric streptavidin-protein conjugation with the tagged proteins. In addition, the functions of a Stav-GOD conjugate, i.e., biotin-binding and glucose oxidase activity, were significantly higher compared to those of streptavidin-GOD conjugates prepared by chemical modification.Copyright © 2011 Elsevier B.V. All rights reserved.