Large-Scale Expression, Refolding, and Purification of the Catalytic Domain of Human Macrophage Metalloelastase (MMP-12) in Escherichia coli Ashfaq A. Parkar,* ,1 Mark D. Stow,† Keith Smith,† Annik K. Panicker,† Jean-Pierre Guilloteau,‡ Raymond Jupp,* and Sarah J. Crowe† *Respiratory and RA Disease Group, Aventi Pharmaceuticals, Route 202-206, Bridgewater, New Jersey 08807-0800; †Dagenham Research Centre, Aventis Pharmaceuticals Ltd., Rainham Road South, Dagenham Essex RM10 7XS, United Kingdom; and ‡Vitry Research Centre, Aventis Pharmaceuticals Ltd., 13 Quai Jules Guesde, 94403 Vitry Sur Seine Cedex, France Received January 10, 2000, and in revised form May 22, 2000 We have cloned, overexpressed, and purified the cat- alytic domain (residues Gly106 to Asn268) of human macrophage metalloelastase (MMP-12) in Escherichia coli. This construct represents a truncated form of the enzyme, lacking the N-terminal propeptide domain and the C-terminal hemopexin-like domain. The over- expressed protein was localized exclusively to insolu- ble inclusion bodies, in which it was present as both an intact form and an N-terminally truncated form. Inclu- sion bodies were solubilized in an 8 M guanidine–HCl buffer and purified by gel filtration chromatography under denaturing conditions. Partial refolding of the protein by dialysis into a 3 M urea buffer caused se- lective degradation of the truncated form of the pro- tein, while the intact catalytic domain was unaffected by proteolysis. An SP–Sepharose chromatography step purified the protein to homogeneity and served also to complete the refolding. The purified protein was homogeneous by mass spectrometry and had an activity similar to that of the recombinant enzyme purified from mammalian cells. The protein was both soluble and monodisperse at a concentration of 9 mg/ ml. This purification procedure enables the produc- tion of 23 mg of protein per liter of E. coli culture and is amenable to large-scale protein production for structural studies. © 2000 Academic Press Extracellular matrix is important as a scaffold for tissue structure and also as an environment regulating cell function and migration in tissues. Matrix metallo- proteinases (MMPs) 2 comprise a family of structurally related enzymes that degrade a variety of extracellular matrix macromolecules, including collagens, proteogly- cans, and elastin (1). Through these activities, MMPs play essential roles in the tissue remodeling that oc- curs during various physiological processes such as normal morphogenesis, wound healing, bone resorp- tion, ovulation, implantation, and uterus involution (2, 3). Physiological and cellular recognition of MMP ac- tivities is not well understood, but a complex array of cellular factors, such as oncogenes, growth factors, hor- mones, and specific protein inhibitors, are involved in controlling expression and latency (4 –7). The abnor- mal expression of MMPs may contribute to destructive processes, including tumor invasiveness, arthritis, ath- erosclerosis, emphysema, pulmonary fibrosis, and tis- sue ulceration (8 –11). The degeneration of connective tissue matrix in these disorders may be brought about by the accumu- lation of inflammatory cells such as macrophages. In pulmonary emphysema, for example, the accumulation of macrophages has been well documented, and the proteolytic enzymes secreted by these macrophages may cause the degradation of elastin fibers and other extracellular matrix components characteristic of this disease (12, 13). Human macrophage metalloelastase (MMP-12) is a potential candidate enzyme for mediat- ing this proteolytic attack since it has a tissue distri- bution that is largely restricted to macrophages and it is capable of degrading elastin as well as various other 1 To whom correspondence should be addressed. Fax: (908) 231- 3052. E-mail: Ash.Parkar@aventis.com. 2 Abbreviations used: MMP, matrix metalloproteinase; MMP-12, human macrophage metalloelastase; IPTG, isopropyl -D-thiogalac- toside; SELDI-MS, surface-enhanced laser desorption ionization mass spectrometry; MMP-12_CAT, catalytic domain of MMP-12. Protein Expression and Purification 20, 152–161 (2000) doi:10.1006/prep.2000.1280, available online at http://www.idealibrary.com on 152 1046-5928/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.