Affinity purification of plasmid DNA directly from crude bacterial cell lysates.
We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with impractically low DNA yields. We have optimized the procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 microg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required.
Darby
Richard A J
RA
School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, United Kingdom.
Forde
Gareth M
GM
Slater
Nigel K H
NK
Hine
Anna V
AV
D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D001419:Bacteria / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods*; D004278:DNA, Superhelical / Q000737:chemistry / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins; D005612:Freeze Drying; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D056968:Lac Repressors; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry
D001426:Bacterial Proteins; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C070802:carboxymethylaspartic acid agarose; D049452:Green Fluorescent Proteins; C033223:polyhistidine; D001224:Aspartic Acid; D007544:Isopropyl Thiogalactoside; D006639:Histidine; D012685:Sepharose; D012259:Ribonuclease, Pancreatic