Purification of miraculin glycoprotein using tandem hydrophobic interaction chromatography

DETAILED DESCRIPTION This invention relates to the use of tandem hydrophobic interaction chromatography (HIC) to facilitate the initial isolation of a plant protein, such as the taste-modifying glycoprotein, Miraculin, from extracts of the pulp of miracle fruits.
The initial purification is accomplished by two (tandem) HIC columns coupled in series such that the first column acts primarily as a guard column to remove most of the polyphenols (defined to include pigments, tannins and other polyphenolic compounds) in plant tissue extracts which are co-extracted with Miraculin.
As a result Miraculin becomes free of plant pigments and is efficiently bound to, or captured on the second HIC column.
The partially purified Miraculin obtained after the HIC step is further purified to homogeneity preferably by ion-exchange and followed by gel filtration chromatography.
Such a separation is therefore based on hydrophobicity, charge, and molecular size.
Tandem HIC eliminates the need for ammonium sulfate fractionation of crude Miraculin extracts, as commonly practiced in the prior art while achieving a purity equivalent to or better than prior art methods.
Ammonium sulfate fractionation involves tedious processing conditions using as much as 50-80% saturated (2-3M) concentrations of ammonium sulfate which leads to yield losses of the saturated active ingredient.
The latter procedure also involves cumbersome operations such as repeated precipitation and centrifugation steps which are expensive to scale-up.
In contrast, the inventive method requires pretreatment of the extract with considerably lower concentrations (as low as 10% or less) of ammonium sulfate before the HIC step.
Moreover, the sample can be applied to the column as soon as it is extracted thus minimizing proteolytic degradation processes.
The inventive method succeeds where previous efforts have been inadequate.
It is robust and fast enough that the entire purification process can be performed at room temperature without loss of activity of Miraculin