Affinity purification of plasmid DNA by temperature-triggered precipitation.
This protocol presents a new method to purify plasmid DNA using temperature-triggered precipitation. The principle is based on the specific DNA-binding affinity of a bacterial metalloregulatory (MerR) protein to its cognate DNA sequence and the temperature responsiveness of elastin-like protein (ELP). A bifunctional ELP-MerR fusion protein is created to enable the precipitation of plasmid DNA, designed to contain the MerR recognition sequence, by a simple temperature trigger. The protocol covers all stages of the process from the design of ELP-MerR fusion proteins and MerR-binding plasmids, to the isolation of plasmid DNA from Escherichia coli cultures after boiling lysis, the subsequent temperature-triggered precipitation of plasmid DNA-fusion protein complexes and final elution of plasmid DNA by mild heating. This protocol is well suited to laboratory research-scale applications, producing plasmid DNA of better purity and similar yield as one of the most commonly used laboratory methods, standard alkaline lysis (known as the midiprep procedure). The protocol takes approximately 30 min to obtain pure plasmid DNA from cell cultures using the temperature-triggered precipitation method.
Lao
U Loi
UL
Department of Chemical and Environmental Engineering, University of California, Riverside, 900 University Ave, Riverside, California 92507, USA.
Kostal
Jan
J
Mulchandani
Ashok
A
Chen
Wilfred
W
D002498:Centrifugation; D004247:DNA / Q000302:isolation & purification*; D004549:Elastin; D004926:Escherichia coli; D005590:Fractional Precipitation; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D011993:Recombinant Fusion Proteins; D004247:DNA; D004549:Elastin