A single step purification for autolytic zinc proteinases

Abstract

We describe a novel single-step method for the purification of stromelysin-1 catalytic domain (SCD) via immobilized metal affinity chromatography under denaturing conditions that inhibit proteolytic activity followed by on-column refolding and spontaneous autolysis of the fusion peptide to yield pure, active stromelysin-1 catalytic domain. The methodology provides a general approach for the rapid purification of large quantities of zinc proteinases.

Figure 1

Autolysis of his₆-fusion peptide with refolding. Lane 1, standards; lane 2, purified SCD eluted prior to refolding; lane 3, eluate after solid phase refolding.

Figure 2

Schematic of solid phase refolding. The protein is purified while denatured (left). Upon refolding, only some of the protein is refolded properly (middle) and the rest is either misfolded or trapped as a protein folding intermediate. Properly refolded enzyme regains activity, autolyzes the fusion peptide tethering it to the resin and elutes from the column.