One-step metal-affinity purification of histidine-tagged proteins by temperature-triggered precipitation.
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.
Stiborova
Hana
H
Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521, USA.
Kostal
Jan
J
Mulchandani
Ashok
A
Chen
Wilfred
W
D000345:Affinity Labels / Q000138:chemical synthesis; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D004549:Elastin / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006639:Histidine / Q000737:chemistry*; D008670:Metals / Q000737:chemistry; D009532:Nickel / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism; D013696:Temperature; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D000345:Affinity Labels; D008670:Metals; D011993:Recombinant Fusion Proteins; D006639:Histidine; D009532:Nickel; D004549:Elastin; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase