Affinity purification of plasmid DNA by temperature-triggered precipitation

作者：Lao U Loi; Kostal Jan; Mulchandani Ashok; Chen Wilfred^*

来源：Nature Protocols, 2007, 2(5): 1263-1268.

DOI：10.1038/nprot.2007.171

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.

• 出版日期2007