The Dac-tag, an affinity tag based on penicillin-binding protein 5

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
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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