Abstract
Hexahistidines are very common tags used in the affinity chromatography purification of recombinant proteins. Although these tags are solely applied for their metal-binding properties, we found that they are also able to perform ester hydrolysis when attached to a protein. For instance, green fluorescent protein (GFP) and the cowpea chlorotic mottle virus (CCMV) are able to perform catalysis after introduction of the His-tag. By attaching a His-tag to an enzyme, a dual-functional catalyst was created, that can perform a two-step cascade reaction. These findings show that the catalytic properties of the hexahistidine tag should be taken into consideration when choosing a suitable protein purification tag.
Highlights
Hexahistidines are very common tags used in the affinity chromatography purification of recombinant proteins
His6-green fluorescent protein (GFP) was dissolved in PBS buffer and the activity of the His-tag was measured by recording the conversion of p-nitrophenol acetate (p-NPA) to p-nitrophenol (p-NP) and acetic acid, using a spectrophotometric
It was found that His6-GFP is able to hydrolyze p-NPA to p-NP, and that its activity can be distinguished from the background hydrolysis of p-NPA (Fig. 1B)
Summary
Hexahistidines are very common tags used in the affinity chromatography purification of recombinant proteins These tags are solely applied for their metal-binding properties, we found that they are able to perform ester hydrolysis when attached to a protein. By attaching a His-tag to an enzyme, a dual-functional catalyst was created, that can perform a two-step cascade reaction These findings show that the catalytic properties of the hexahistidine tag should be taken into consideration when choosing a suitable protein purification tag. We reasoned that the introduction of a His-tag in a protein for purification purposes might not be as innocent as previously thought Since histidines have such unique properties, as a consequence of their side chain imidazole functionalities, the introduction of six copies of this amino acid could have serious implications on the properties of the resulting tagged protein. Our findings show that the catalytic properties of His-tags should be taken into consideration, in addition to their advantageous properties, when choosing a protein purification tag for a specific application
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