Functional human insulin-degrading enzyme can be expressed in bacteria

Insulin-degrading enzyme (IDE) has been shown to degrade a number of biolog ically important peptides, including insulin and the amyloid-beta protein i mplicated in Alzheimer's disease. However, lack of a facile method to gener ate purified enzyme and related mutants has made it difficult to study the precise role of IDE in the clearance of these peptides, Therefore, we deter mined whether recombinant wild-type and mutant human IDEs can be overexpres sed as functional enzymes in bacteria. Three vectors carrying cDNAs encodin g N-terminally polyhistidine-tagged recombinant IDEs were constructed, and the proteins expressed in Escherichia coli were purified by metal affinity chromatography (final yield approximate to 8 mg per liter of culture). The recombinant IDEs, Like the endogenous mammalian enzyme, migrate with 110-kD a apparent molecular masses in. SDS-polyacrylamide gels and as a approximat e to 200-kDa species in gel filtration. Further analysis by native PAGE ind icates that IDE can form multimers of different complexities. The wild-type recombinant endopeptidase degrades insulin, with an efficiency similar to that of the enzyme purified from mammalian tissues. Purified IDEs are stabl e at 4 degrees C for at least 1 month. Purified recombinant protein, was us ed to raise specific polyclonal antibodies that can immunoprecipitate nativ e mammalian IDE, Thus, the procedure described allows the rapid production of large amounts of purified IDE and demonstrates that IDE can be produced in an active form in the absence of other potential interacting mammalian p roteins. (C) 2000 Academic Press.