Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker.
The sequence-specific affinity chromatographic isolation of plasmid DNA from crude lysates of E. coli DH5alpha fermentations is addressed. A zinc finger-GST fusion protein that binds a synthetic oligonucleotide cassette containing the appropriate DNA recognition sequence is described. This cassette was inserted into the SmaI site of pUC19 to enable the affinity isolation of the plasmid. It is shown that zinc finger-GST fusion proteins can bind both their DNA recognition sequence and a glutathione-derivatized solid support simultaneously. Furthermore, a simple procedure for the isolation of such plasmids from clarified cell lysates is demonstrated. Cell lysates were clarified by cross-flow Dean vortex microfiltration, and the permeate was incubated with zinc finger-GST fusion protein. The resulting complex was adsorbed directly onto glutathione-Sepharose. Analysis of the glutathione-eluted complex showed that plasmid DNA had been recovered, largely free from contamination by genomic DNA or bacterial cell proteins.
School of Engineering and Applied Science, Aston University, Aston Triangle, Birmingham, United Kingdom.
John
J
Woodgate
David
D
Palfrey
David A
DA
Nagel
Anna V
AV
Hine
Nigel K H
NK
Slater
D002846:Chromatography, Affinity / Q000379:methods*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000378:metabolism*; D008957:Models, Genetic; D010865:Pilot Projects; D010957:Plasmids / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D014462:Ultrafiltration / Q000379:methods*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase