Large-scale expression, refolding, and purification of the catalytic domain of human macrophage metalloelastase (MMP-12) in Escherichia coli.
We have cloned, overexpressed, and purified the catalytic 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 overexpressed protein was localized exclusively to insoluble inclusion bodies, in which it was present as both an intact form and an N-terminally truncated form. Inclusion 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 selective degradation of the truncated form of the protein, 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 production of 23 mg of protein per liter of E. coli culture and is amenable to large-scale protein production for structural studies.
Parkar
A A
AA
Respiratory and RA Disease Group, Aventi Pharmaceuticals, Bridgewater, New Jersey 08807-0800, USA. Ash.Parkar@aventis.com
Stow
M D
MD
Smith
K
K
Panicker
A K
AK
Guilloteau
J P
JP
Jupp
R
R
Crowe
S J
SJ
D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli; D005470:Fluorometry; D006801:Humans; D002479:Inclusion Bodies; D008027:Light; D008264:Macrophages / Q000201:enzymology*; D013058:Mass Spectrometry; D053508:Matrix Metalloproteinase 12; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012542:Scattering, Radiation; D012995:Solubility; D014508:Urea / Q000494:pharmacology; D000515:alpha 1-Antitrypsin / Q000378:metabolism
D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D014508:Urea; D008666:Metalloendopeptidases; C507122:MMP12 protein, human; D053508:Matrix Metalloproteinase 12