The Dac-tag, an affinity tag based on penicillin-binding protein 5
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- 作者：David Wei Lee ; Mark Peggie ; Maria Deak ; Rachel Toth ; Zoe Olivia Gage ; Nicola Wood ; Christina Schilde ; Thimo Kurz ; Axel Knebel
- 关键词：Affinity tag ; Protein purification ; Penicillin-binding protein 5 ; Ampicillin Sepharose
- 刊名：Analytical Biochemistry
- 出版年：2012
- 出版时间：1 September, 2012
- 年：2012
- 卷：428
- 期：1
- 页码：64-72
- 全文大小：661 K
文摘
Penicillin-binding protein 5 (PBP5), a product of the Escherichia coli gene dacA, possesses some ¦Â-lactamase activity. On binding to penicillin or related antibiotics via an ester bond, it deacylates and destroys them functionally by opening the ¦Â-lactam ring. This process takes several minutes. We exploited this process and showed that a fragment of PBP5 can be used as a reversible and monomeric affinity tag. At ambient temperature (e.g., 22 ¡ãC), a PBP5 fragment binds rapidly and specifically to ampicillin Sepharose. Release can be facilitated either by eluting with 10 mM ampicillin or in a ligand-free manner by incubation in the cold (1-10 ¡ãC) in the presence of 5 % glycerol. The ¡°Dac-tag¡±, named with reference to the gene dacA, allows the isolation of remarkably pure fusion protein from a wide variety of expression systems, including (in particular) eukaryotic expression systems.