The Dac-tag, an affinity tag based on penicillin-binding protein 5.
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 10mM 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.
Advantagen, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK.
David Wei
DW
Lee
Mark
M
Peggie
Maria
M
Deak
Rachel
R
Toth
Zoe Olivia
ZO
Gage
Nicola
N
Wood
Christina
C
Schilde
Thimo
T
Kurz
Axel
A
Knebel
D000818:Animals; D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods*; D004023:Dictyostelium / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007313:Insecta; D046915:Penicillin-Binding Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D002021:Buffers; D029968:Escherichia coli Proteins; C569173:PBP5 protein, E coli; D046915:Penicillin-Binding Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins