One‐step metal‐affinity purification of histidine‐tagged proteins by temperature‐triggered precipitation

Abstract

The feature of elastin-like proteins (ELPs) to reversibly precipitate above their transition temperature was exploited as a general method for the purification of histidine (His)-tagged proteins. The principle of the single-step metal-affinity method is based on coordinated ligand-bridging between the modified ELPs and the target proteins. ELPs with repeating sequences of [(VPGVG)(2)(VPGKG)(VPGVG)(2)](21) were synthesized and the free amino groups on the lysine residues were modified by reacting with imidazole-2-carboxyaldehyde to incorporate the metal-binding ligands into the ELP bio- polymers. Biopolymers charged with Ni(2+) were able to interact with a His tag on the target proteins based on metal coordination chemistry. Purifications of two His-tagged enzymes, beta-D-galactosidase and chloramphenicol acetyltransferase, were used to demonstrate the utility of this general method and over 85% recovery was observed in both cases. The bound enzymes were easily released by addition of either EDTA or imidazole. The recovered ELPs were reused four times with no observable decrease in the purification performance.

Peptide-based biopolymers in biomedicine and biotechnology

“…ELP-tagged proteins A, G, and L provide a general platform to capture antibody molecules of different subclasses [83] and allow complex multiplexing of protein purification and detection schemes. ELP-nickel conjugates can be used successfully to isolate proteins tagged with a short histidine sequence [84]. To purify unmodified proteins, ELP-tagged singlechain antibodies can be selectively bound to their protein partners.…”

Purification of recombinant proteins from Escherichia coli at low expression levels by inverse transition cycling

“…2), and the amounts of the recovered proteins are in the picomole range from a liter of culture, which provides enough protein for downstream analysis by mass spectrometry [21][22][23]. Furthermore, because of the detection limits of scintillation counting for 14 C radioactivity, we were unable to test the lower limits of this technique, but we see no inherent limitations in purifying even lower concentrations of ELP fusion proteins by this methodology.…”

“…A number of different proteins have been purified by this method using either centrifugation [11][12][13][14][15][16][17][18] or micro-filtration [19]. The direct purification of ELPs has also been extended to the capture of native proteins by ELP-tagged capture reagents [14][15][16][17].Proteins expressed at the level of micrograms per liter of culture are difficult to purify by chromatography because of the losses associated with non-specific and irreversible adsorption of the target protein to chromatography resins as well as the relative increase in contamination from host proteins that are non-specifically adsorbed to the chromatography resin are subsequently eluted with the target protein. We demonstrate a new variant of ITC that solves *Corresponding Author.…”

“…A number of different proteins have been purified by this method using either centrifugation [11][12][13][14][15][16][17][18] or micro-filtration [19]. The direct purification of ELPs has also been extended to the capture of native proteins by ELP-tagged capture reagents [14][15][16][17].…”

Affinity purification of plasmid DNA by temperature-triggered precipitation

“…ELPs are artificial peptides consisting of repeating VPGVG that have been shown to undergo a reversible phase transition upon environmental stimuli from watersoluble forms into hydrophobic aggregates, facilitating recovery by precipitation 13 . Advances in genetic engineering have made it possible to fuse different protein partners to ELP while preserving the phase transition and the partners' functionalities [14][15][16] . The use of ELP fusion proteins for affinity precipitation has been demonstrated during the purification of a wide range of ELP-fusion proteins, His-tagged proteins and antibodies [14][15][16][17] .…”