Purification of Recombinant Green Fluorescent Protein Using Chromatofocusing with a pH Gradient Composed of Multiple Stepwise Fronts

Abstract

Green fluorescent protein (GFP), which fluoresces in the green region of the visible spectrum and is widely used as a reporter for gene expression and regulation, was overexpressed in the JM105 strain of Escherichia coli transformed with pBAD-GFP. A two-step chromatofocusing procedure was used to purify GFP starting from cell lysate, with each step employing a pH gradient extending from pH 5.5 to 4.0. The first chromatofocusing step was performed using a low-pressure column in which a retained stepwise pH front formed by adsorbed buffering species was used to capture GFP directly from clarified cell lysate and selectively focus it into a chromatographic band. The second step utilized a high-performance column under mass overloaded conditions where a similar pH front acted as a protein displacer and led to the formation of a highly concentrated rectangular band of GFP. The overall procedure yielded a 50-fold increase in purity, a 20-fold volume reduction, and a recovery and purity for GFP of 60% and 80%, respectively. Because the method employs a strong-base ion-exchange column packing and low-cost buffers formed with formic and acetic acids instead of the proprietary column packings and polyampholyte elution buffers more generally used for chromatofocusing, it appears to be a practical alternative for the preparative ion-exchange chromatography of GFP in particular and for the recovery of recombinant proteins from cell lysate in general. A discussion is also given concerning the choice of appropriate buffers for the rational design of pH gradients involving retained, stepwise pH fronts that span a given pH range and of the use of the fluorescence properties of GFP for flow visualization and chromatographic process development.

Purification of recombinant green fluorescent protein by three-phase partitioning

“…4) of these two purified preparations obtained under the conditions of crude:tert-butanol ratios of 1:1 and 1:2 showed that both preparations are comparable in levels of purity. The estimated molecular mass (SDS-PAGE) was found to be 27 000 Da which agreed well with the reported molecular mass of the protein [4]. Thus any of the two procedures utilizing the two cycles of TPP constitute a fairly simple and efficient purification process for GFP.…”

“…Yakhnin et al [3] described a four-step purification procedure with rather poor 36% yield. Narahari et al [4] used two chromatofocussing steps to obtain a 50-fold increase in purity with 60% recovery. Use of immobilized metal affinity chromatography by Li et al [5] is perhaps the most convenient procedure.…”

Mild and cost-effective green fluorescent protein purification employing small synthetic ligands

“…Purification processes for GFP fusion proteins require multiple steps of chromatography or immunoaffinity separations [10][11][12][13][14][15][16]. Multi-step purification protocols involve the use of different techniques, including hydrophobic-interaction chromatography (HIC) [13], immobilized metal affinity chromatography (IMAC) [11], and aqueous two-phase extraction (ATPE) [12].…”

Purification of recombinant GFP produced by Agrobacterium-mediated transient expression in Nicotiana excelsior

“…It includes tag-based protocols [8,9], which does not always allow the separation of improperly folded or cyclized GFP from the correctly folded form of this protein [10], size exclusion chromatography [11], chromatofocusing [12], organic extraction [11], etc. It includes tag-based protocols [8,9], which does not always allow the separation of improperly folded or cyclized GFP from the correctly folded form of this protein [10], size exclusion chromatography [11], chromatofocusing [12], organic extraction [11], etc.…”