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Purification of rIL-13 Using an UNO Q1 Column

Interleukin 13 is a recently described cytokine produced by activated T cells. This protein is responsible for both positive and negative responses to regulatory molecules within the immune system. rIL-13 has been shown to modulate macrophage functions as well as play a direct role in regulation of proliferation and differentiation of hematopoietic stem cells. In addition, a recent study has indicated that rIL-13 inhibits human immunodeficiency virus type I production in blood derived macrophages. For this application, recombinant rIL-13 was overexpressed in E. coli. in the form of inclusion bodies. This report describes the isolation and purification of rIL-13 using an UNO Q1 (Bio-Rad catalog # 720-0001) column.

rIL-13 was obtained from the cell pellet in a two part process, sample preparation and chromatographic separation. See Figure 1 for a flow chart of sample preparation. Sample preparation involved suspending the cell pellet in 50 mM Tris, pH 8.0, 20 mM DTT with 350 l of bacterial protease inhibitor (Sigma P8465). The suspension was then passed through a Microfluidics cell disruptor at 12,000 psi five times. The lysed cell suspension was then centrifuged, washed, and the rIL-13 was solubilized with 8 M urea. It was found that rIL-13 will only solubilize with a high concentration of chaotrope under reducing conditions. The final solubilized preparation was then filtered through a 0.45 m filter and applied to an UNO Q1 column.

The rIL-13 was eluted by a linear gradient as described in Figure 2. Due to the viscosity of the buffers used in the experiment, the UNO Q1 column was run at 3 ml/min.

Figure 2 shows the elution profile from the UNO Q1 column. rIL-13 was found to elute from this column in the conductivity range of 1.75-3.55 ms/cm.

SDS-PAGE (Figure 3) analysis using a 15% Tris-HCl Ready Gel (Bio-Rad catalog #161-1157) indicated fractions 1820 contained rIL-13 of at least 90% purity, while fraction 21 contained a small amount of impurities. A western blot of the gel was performed to identify the rIL-13 (data not shown). The apparent heterogeneity in the elution profile may arise from protein-protein interaction or folding intermediates, since by SDS-PAGE, the rIL-13 was nearly homogeneous.

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We gratefully acknowledge receipt of rIL-13 containing E. coli cell pellet constructed by Madeleine Huey working under Dr. John Roberts at UCSF liver transplant.

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