Purification of Recombinant Green Fluorescent Protein Using Chromatofocusing with a pH Gradient Composed of Multiple Stepwise Fronts
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- 作者：Chittoor R. Narahari ; Lisa Randers-Eichhorn ; John C. Strong ; Natraj Ramasubramanyan ; Govind Rao ; and Douglas D. Frey
- 刊名：Biotechnology Progress
- 出版年：2001
- 出版时间：February 2001
- 年：2001
- 卷：17
- 期：1
- 页码：150 - 160
- 全文大小：155K
- 年卷期：v.17,no.1(February 2001)
- ISSN：1520-6033
文摘
Green fluorescent protein (GFP), which fluoresces in the green region of the visiblespectrum and is widely used as a reporter for gene expression and regulation, wasoverexpressed in the JM105 strain of Escherichia coli transformed with pBAD-GFP.A two-step chromatofocusing procedure was used to purify GFP starting from celllysate, with each step employing a pH gradient extending from pH 5.5 to 4.0. Thefirst chromatofocusing step was performed using a low-pressure column in which aretained stepwise pH front formed by adsorbed buffering species was used to captureGFP directly from clarified cell lysate and selectively focus it into a chromatographicband. The second step utilized a high-performance column under mass overloadedconditions where a similar pH front acted as a protein displacer and led to theformation of a highly concentrated rectangular band of GFP. The overall procedureyielded a 50-fold increase in purity, a 20-fold volume reduction, and a recovery andpurity 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 aceticacids instead of the proprietary column packings and polyampholyte elution buffersmore generally used for chromatofocusing, it appears to be a practical alternative forthe preparative ion-exchange chromatography of GFP in particular and for the recoveryof recombinant proteins from cell lysate in general. A discussion is also givenconcerning the choice of appropriate buffers for the rational design of pH gradientsinvolving retained, stepwise pH fronts that span a given pH range and of the use ofthe fluorescence properties of GFP for flow visualization and chromatographic processdevelopment.