Affinity purification of plasmid DNA by temperature-triggered precipitation

Abstract

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 temperaturetriggered precipitation method.

De Novo Design of Supercharged, Unfolded Protein Polymers, and Their Assembly into Supramolecular Aggregates

“…In view of that, the results reported in Figure 3 indicate that a hollow and stable wall remains when the inorganic core is dissolved. Compared to capsules formed by standard synthetic polyelectrolytes, such as polystyrene sulfonate (PSS) and poly(allylamine hydrochloride) (PAH), [18] the walls of capsules based on supercharged proteins are rather porous (see Supporting Information, Figure S10, [12][13]. This may be a result of the lower charge density of E57 and K48 compared to PSS and PAH (see Supporting Information, Table 1), which results in a higher mechanical pressure during the dissolution procedure.…”

“…[12] For tissue engineering purposes, ELPs were designed as thermally sensitive hydrogels that solidify when injected into the body. [13] Furthermore, their temperature responsiveness was utilized for drug delivery applications.…”

Elastomeric Polypeptides

“…The third purification procedure is based on the combination of temperature-triggered aggregation and affinity capture and has been used to not only purify proteins [50-54] but also other molecules [55, 56], and for the removal of pollutants from a solution [57-59]. For this procedure, ELP is conjugated to a capture reagent, which can be done either genetically or chemically.…”

“…A schematic overview of the plasmid purification via temperature-triggered affinity capture is depicted in Fig. 11 [55, 56]. …”

“…Subsequently, the plasmid is eluted from the DNA binding protein by mild heating, after which the ELP-DNA binding protein is removed and recycled via centrifugation to obtain the purified plasmid in solution. Adapted from [56] by permission from Macmillan Publishers Ltd: Nature Protocols, copyright 2007…”