Affinity purification of plasmid DNA by temperature-triggered precipitation.
This report describes a new plasmid DNA purification method, which takes advantage of the DNA-binding affinity and specificity of the bacterial metalloregulatory protein MerR, and of the temperature responsiveness of elastin-like proteins (ELPs). Upon increasing the temperature, ELP undergoes a reversible phase transition from water-soluble forms into aggregates, and this property was exploited for the precipitation of plasmid DNA containing the MerR recognition sequence by a simple temperature trigger. In one purification step, plasmid DNA was purified from E. coli cell lysates to a better purity than that prepared by a standard alkaline purification method, with no contaminating chromosomal DNA and cellular proteins. This protein-based approach, in combination with the reversible phase transition feature of ELP, makes the outlined method a promising candidate for large-scale purification of plasmid DNA for sensitive applications such as nonviral gene therapy or DNA vaccines.
Kostal
Jan
J
Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521, USA.
Mulchandani
Ashok
A
Chen
Wilfred
W
D011232:Chemical Precipitation; D002846:Chromatography, Affinity / Q000379:methods*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004549:Elastin / Q000031:analogs & derivatives / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry* / Q000302:isolation & purification*; D011485:Protein Binding; D013696:Temperature
D004268:DNA-Binding Proteins; D004549:Elastin