Abstract:
:The membrane-anchored metalloproteinase ADAM17 (TNF-alpha converting enzyme; TACE; EC 3.4.24.86) continues to be an attractive drug target in inflammatory diseases and cancer. Cocrystallization of its catalytic domain with a lead compound was complicated by the tenacious retention of the prodomain that has been shown to be enhanced if ADAM17 is expressed without the disintegrin/cysteine-rich domain that normally follows the N-terminal metalloproteinase. When a truncated form of ADAM17 composed of the signal peptide with the pro- and catalytic domains was expressed in baculovirus-infected insect cells, the major secreted product was a ternary complex of two prodomain fragments with the catalytic domain. The component polypeptides of the ternary complex were characterized by N-terminal analysis and mass spectrometry. Internal cleavage of the propeptide occurred following Arg-58, and a carboxypeptidase variably removed up to three basic residues from the newly created C-terminus. Cleavage at the C-terminus of the propeptide occurred after Arg-214. To prepare ADAM17 for crystal growth, a drug-like inhibitor was used to displace the propeptide and the complex of the catalytic domain with the inhibitor was isolated by size-exclusion chromatography and crystallized.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Hoth LR,Tan DH,Wang IK,Wengender PA,Thompson MA,Kamath AV,Geoghegan KFdoi
10.1016/j.pep.2006.10.021subject
Has Abstractpub_date
2007-04-01 00:00:00pages
313-9issue
2eissn
1046-5928issn
1096-0279pii
S1046-5928(06)00335-4journal_volume
52pub_type
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