Development of an efficiently cleaved, bioactive, highly pure FLAG-tagged recombinant human Mullerian Inhibiting Substance.
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.
Papakostas
Thanos D
TD
Angiogenesis Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 325 Cambridge St., Boston, MA, USA.
Pieretti-Vanmarcke
Rafael
R
Nicolaou
Fotini
F
Thanos
Aristomenis
A
Trichonas
George
G
Koufomichali
Xanthi
X
Anago
Kosisochukwu
K
Donahoe
Patricia K
PK
Teixeira
Jose
J
MacLaughlin
David T
DT
Vavvas
Demetrios
D
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
D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D054304:Anti-Mullerian Hormone; C085706:FLAG peptide