PURIFICATION OF RECOMBINANT HISTIDINE-TAGGED CATALYTIC DOMAIN OF MMP-13 IN ONE-STEP USING AFFINITY COLUMN AND RENATURATION OF IT WITH HISTIDINE-TAG

Abstract Collagenase-3 (MMP-13) is a member of the matrix metalloproteinases (MMPs), which are closely related to a range of pathologies. The one-step purification of histidine-tagged (His-tagged) recombinant catalytic domain of collagenase-3 (His-cdMMP-13) expressed in Escherichia coli by Ni (II) immobilized metal ion affinity chromatography (IMAC) was investigated. Four factors, including the concentration of urea and NaCl in the binding buffer, flow rate, and loaded quantity, influencing the purity and recovery yield of the target protein were investigated in details. Under the optimal condition, binding buffer with 15 mM imidazole, a flow rate of 1.0 mL/min and 1.2 mL (22.7 mg) sample loaded, His-cdMMP-13 with purity of 98% and recovery yield of 57% was obtained. Further study showed that the adding of NaCl into binding buffer obviously improved the recovery yield to 71%, but decreased the purity to 91%. Finally, the activity of His-cdMMP-13 with His-tag could be renatured after refolding by gel chromatography. Keywords: His-tagimmobilized metal ion affinity chromatographyinclusion bodiesMMP-13 catalytic domainpurificationrefolding ACKNOWLEDGMENT The authors are grateful to Professor Xuexun Fang for providing the E. coli strain BL21 to express the His-tagged MMP-13 catalytic domain. Notes a Besides NaCl, all binding buffers contain 6 M urea, 15 mM imidazole, and 20 mM Tris-HCl (pH 7.5) with flow rate of 1.0 mL/min and loaded sample quantity of 1.2 mL (19 mg/mL).