Purification of the proprotein convertase furin by affinity chromatography based on PC-specific inhibitors

Abstract

In eucaryotes, many secreted proteins and peptides are proteolytically excised from larger precursor proteins by a specific class of serine proteases, the proprotein/prohormone convertases (PCs). This cleavage is essential for substrate activation, making the PCs very interesting pharmacological targets in cancer and infectious disease research. Correspondingly, their structure, function and inhibition are intensely studied - studies that require the respective target proteins in large amounts and at high purity. Here we describe the development of a novel purification protocol of furin, the best-studied member of the PC family. We combined the heterologous expression of furin from CHO cells with a novel purification scheme employing an affinity step that efficiently extracts only active furin from the conditioned medium by using furin-specific inhibitor moieties as bait. Several potential affinity tags were synthesized and their binding to furin characterized. The best compound, Biotin-(Adoa)(2)-Arg-Pro-Arg-4-Amba coupled to streptavidin-Sepharose beads, was used in a three-step chromatographic protocol and routinely resulted in a high yield of a homogeneous furin preparation with a specific activity of ~60 units/mg protein. This purification and the general strategy can easily be adapted to the efficient purification of other PC family members.

Figure 1

Chemical structure of Biotin-(Adoa)₂-Arg-Pro-Arg-4-Amba (inhibitor 6) for final use in the affinity-based purification.

Figure 2. Qualitative binding assay of furin to different affinity tags: SDS-PAGE of inhibitor 6 and negative control

(A) Negative control. Strong furin bands are visible for the applied material (A) and the supernatant (S) showing protein that does not bind to the beads. No protein can be observed in the fractions of the elution steps with 150–1000 mM NaCl, indicated by 150, 250, 500 and 1000, and in the pellet (P). (B) Qualitative binding assay of inhibitor 6. Furin is seen in all fractions except for the pellet; the strongest bands are visible for the applied material (A) and the elution steps at 500 mM and 1 M NaCl. ‘M’ indicates the molecular weight marker; the respective weights are given on the left-hand side in kDa. Each lane corresponds to 40 μl of the respective fractions.

Figure 3. Chromatographic purification of mouse furin

The absorbance at 280 nm (black line, left ordinate) and the relative furin activity of selected fractions (green bars) is shown as function of total volume of the chromatography and the sample application step (F) is indicated in panels (A) and (B). (A) DEAE-chromatography; (B) affinity chromatography on Biotin-(Adoa)2-Arg-Pro-Arg-4-Amba coupled to streptavidin-Sepharose showing in addition the applied salt gradient in orange; and (C): size exclusion chromatography. In panel (B), the first fraction of the protein elution applied to the SDS-PAGE (Figure 4) is indicated with (1).

Figure 4. SDS-PAGE of affinity chromatography

Selected fractions of the affinity chromatography step were analyzed by SDS-PAGE. ‘A’ indicates the applied material, ‘F1’–‘F3’ the fractions of the flow-through, ‘W’ the washing step, ‘M’ the molecular mass markers (the respective weights are given in kDa) and lanes 1–10 show the peak elution fractions. The arrows on both sides mark the furin band.

Figure 5. Purification overview

The conditioned medium and pool fraction of the different purification steps were analyzed by SDS-PAGE. The lanes from left to right show the molecular weight marker (M; the respective weights are given on the left-hand side in kDa), the conditioned medium (Med), the ion exchange chromatography pool (IEC), the affinity chromatography pool (AC) and the size exclusion chromatography pool (SEC), containing 2.5 μg total protein each. The arrow marks the furin band.