Purification of recombinant green fluorescent protein using chromatofocusing with a pH gradient composed of multiple stepwise fronts.
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.
Narahari
C R
CR
Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, Baltimore, Maryland 21250, USA.
Randers-Eichhorn
L
L
Strong
J C
JC
Ramasubramanyan
N
N
Rao
G
G
Frey
D D
DD
D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins