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We have studied the equilibrium uptake behavior and mass transfer rate of recombinant apolipoprotein A-I Milano (apo A-I M ) on Q Sepharose HP under non-denaturing, partially denaturing, and fully denaturing conditions. The protein of interest in this study is composed of amphipathic α helices that serve to solubilize and transport lipids. The dual nature of this molecule leads to the formation of micellar-like structures and self association in solution. Under non-denaturing conditions equilibrium uptake is 134 mg/mL media and the isotherm is essentially rectangular. When fully denatured with 6 M urea, the equilibrium binding capacity decreases to 25 mg/mL media and the isotherm becomes less favorable. The decrease in both binding affinity and media capacity when the protein is completely denatured with 6 M urea can be explained by the loss of all alpha helical structure. The rate of apo A-I M mass transfer on Q Sepharose HP was characterized using a macropore diffusion model. Results of modeling studies indicate that effective pore diffusivity increases from 4.5 × 10 -9 cm 2 /s in the absence of urea to 6.0 × 10 -8 cm 2 /s when apo A-I M is fully denatured with 6 M urea. Based on light-scattering data reported for apo A-I, protein self association appears to be the dominant cause of slow protein mass transfer observed under non-denaturing conditions.

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.