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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