title: Functional human insulin-degrading enzyme can be expressed in bacteria.
abstract: Insulin-degrading enzyme (IDE) has been shown to degrade a number of biologically important peptides, including insulin and the amyloid-beta protein implicated in Alzheimer's disease. However, lack of a facile method to generate purified enzyme and related mutants has made it difficult to study the precise role of IDE in the clearance of these peptides. Therefore, we determined whether recombinant wild-type and mutant human IDEs can be overexpressed as functional enzymes in bacteria. Three vectors carrying cDNAs encoding N-terminally polyhistidine-tagged recombinant IDEs were constructed, and the proteins expressed in Escherichia coli were purified by metal affinity chromatography (final yield approximately 8 mg per liter of culture). The recombinant IDEs, like the endogenous mammalian enzyme, migrate with 110-kDa apparent molecular masses in SDS-polyacrylamide gels and as a approximately 200-kDa species in gel filtration. Further analysis by native PAGE indicates 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 stable at 4 degrees C for at least 1 month. Purified recombinant protein was used to raise specific polyclonal antibodies that can immunoprecipitate native 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 proteins.
authors.affiliation: Ben May Institute for Cancer Research, University of Chicago, Illinois 60637, USA.
authors.forename: V
authors.initials: V
authors.lastname: Chesneau
authors.forename: M R
authors.initials: MR
authors.lastname: Rosner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007328:Insulin / Q000737:chemistry*; D007339:Insulysin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D007328:Insulin; D011993:Recombinant Fusion Proteins; D006639:Histidine; D007339:Insulysin