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

TY - JOUR

T1 - The Dac-tag, an affinity tag based on penicillin-binding protein 5

AU - Lee, David Wei

AU - Peggie, Mark

AU - Deak, Maria

AU - Toth, Rachel

AU - Gage, Zoe Olivia

AU - Wood, Nicola

AU - Schilde, Christina

AU - Kurz, Thimo

AU - Knebel, Axel

N1 - Copyright 2012 Elsevier B.V., All rights reserved.

PY - 2012

Y1 - 2012

N2 - Penicillin-binding protein 5 (PBP5), a product of the Escherichia coli gene dacA, possesses some ß-lactamase activity. On binding to penicillin or related antibiotics via an ester bond, it deacylates and destroys them functionally by opening the ß-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.

AB - Penicillin-binding protein 5 (PBP5), a product of the Escherichia coli gene dacA, possesses some ß-lactamase activity. On binding to penicillin or related antibiotics via an ester bond, it deacylates and destroys them functionally by opening the ß-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.

UR - https://www.scopus.com/pages/publications/84864037626

U2 - 10.1016/j.ab.2012.06.007

DO - 10.1016/j.ab.2012.06.007

M3 - Article

C2 - 22705378

AN - SCOPUS:84864037626

SN - 0003-2697

VL - 428

SP - 64

EP - 72

JO - Analytical Biochemistry

JF - Analytical Biochemistry

IS - 1

ER -