Lee, D. W., Peggie, M., Deak, M., Toth, R., Gage, Z. O., Wood, N., Schilde, C., Kurz, T. and Knebel, A. (2012) The Dac-tag, an affinity tag based on penicillin-binding protein 5. Analytical Biochemistry, 428(1), pp. 64-72. (doi: 10.1016/j.ab.2012.06.007) (PMID:22705378)
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Abstract
Penicillin-binding protein 5 (PBP5), a product of the Escherichia coli gene dacA, possesses some β-lactamase activity. On binding to penicillin or related antibiotics via an ester bond, it deacylates and destroys them functionally by opening the β-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 °C), 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 °C) 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.
Item Type: | Articles |
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Status: | Published |
Refereed: | Yes |
Glasgow Author(s) Enlighten ID: | Kurz, Dr Thimo |
Authors: | Lee, D. W., Peggie, M., Deak, M., Toth, R., Gage, Z. O., Wood, N., Schilde, C., Kurz, T., and Knebel, A. |
College/School: | College of Medical Veterinary and Life Sciences > School of Molecular Biosciences |
Journal Name: | Analytical Biochemistry |
Publisher: | Elsevier |
ISSN: | 0003-2697 |
ISSN (Online): | 1096-0309 |
Published Online: | 15 June 2012 |
Copyright Holders: | Copyright © 2012 Elsevier Inc. |
First Published: | First published in Analytical Biochemistry 428: 64-72 |
Publisher Policy: | Reproduced under a Creative Commons License |
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