title: Development of an efficiently cleaved, bioactive, highly pure FLAG-tagged recombinant human Mullerian Inhibiting Substance.
abstract: Mullerian Inhibiting Substance (MIS), a member of the TGF-beta family, causes regression of the Mullerian duct in male embryos, after binding to Mullerian Inhibiting Substance Receptor II (MISRII). It has also been extensively demonstrated that it can inhibit proliferation of various cancer cell lines such as ovarian, prostate, and breast cancer in vitro and in vivo. Hence, the availability of a recombinant, epitope tagged, bioactive MIS is important for the selection of patients for treatment and for probing novel molecular targets for MIS in various tissues. To this end, we have expressed a recombinant, internally FLAG-tagged form of hMIS with the tag (DYKDDDDK) immediately after the cleavage site (427-428) of MIS at the C-terminus with a modified dibasic cleavage motif sequence. We show that this construct results in a highly pure, endogenously processed (cleaved) FLAG MIS, that causes complete regression of the Mullerian Duct in an organ culture assay. In addition, purified FLAG MIS was able to bind and affinity purify both transfected and endogenous MIS type II receptor. The availability of this fully functional, epitope tagged form of MIS should facilitate scale-up for preclinical and clinical use and should also be used for the study of MIS binding proteins and for tracking in pharmacokinetic studies.
authors.affiliation: Angiogenesis Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 325 Cambridge St., Boston, MA, USA.
authors.forename: Thanos D
authors.initials: TD
authors.lastname: Papakostas
authors.forename: Rafael
authors.initials: R
authors.lastname: Pieretti-Vanmarcke
authors.forename: Fotini
authors.initials: F
authors.lastname: Nicolaou
authors.forename: Aristomenis
authors.initials: A
authors.lastname: Thanos
authors.forename: George
authors.initials: G
authors.lastname: Trichonas
authors.forename: Xanthi
authors.initials: X
authors.lastname: Koufomichali
authors.forename: Kosisochukwu
authors.initials: K
authors.lastname: Anago
authors.forename: Patricia K
authors.initials: PK
authors.lastname: Donahoe
authors.forename: Jose
authors.initials: J
authors.lastname: Teixeira
authors.forename: David T
authors.initials: DT
authors.lastname: MacLaughlin
authors.forename: Demetrios
authors.initials: D
authors.lastname: Vavvas
mesh_terms: D000595:Amino Acid Sequence; D054304:Anti-Mullerian Hormone / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D054304:Anti-Mullerian Hormone; C085706:FLAG peptide