Refolding of Recombinant Histidine-Tagged Catalytic Domain of MMP-13 from Escherichia coli with Ion-Exchange Chromatography for Higher Bioactivity

Abstract

The development of inhibitors and medicines for collagenase-3 (MMP-13) as a therapeutic target for the treatment of some diseases such as cancer and arthritis has attracted much interest. A key technology for screening inhibitors and medicines is to obtain the activated recombinant catalytic domain of collagenase-3 (cdMMP-13). In this study, for the first time, a chromatographic method was used to refold the Histidine-tagged (His-tagged) cdMMP-13 (His-cdMMP-13). After the optimization of gradient elution modes and chromatographic conditions, the recovery yield of His-cdMMP-13 reached ∼83% with a relative activity of ∼92%. Both the recovery yield and relative activity of the His-cdMMP-13 were higher than those obtained by dialysis. Finally, to prevent the slight autoproteolytic degradation in the desalting step, different small molecule inhibitors were separately added into the desalting buffer, and the results of circular dichroism (CD) spectral analysis indicate that the addition of inhibitors did not affect the secondary structure of the target protein. The His-cdMMP-13, refolded through chromatography with a good activity, may be used to screen inhibitors and medicines.

Keywords:

• anion-exchange chromatography
• circular dichroism spectral
• degradation
• Histidine-tagged MMP-13 catalytic domain
• protein refolding
• size-exclusion chromatography

Acknowledgments

The authors are grateful to Xuexun Fang for providing the E. coli strain BL21 to express the His-tagged MMP-13 catalytic domain.

Notes

^a Relative activity = activity of target protein/activity of commercial reference × 100%.