title: Isolation and characterization of the nuclear serpin MENT.
abstract: A balance between proteolytic activity and protease inhibition is required to maintain the appropriate function of biological systems in which proteases play a role. The Myeloid and Erythroid Nuclear Termination protein, MENT, is a nonhistone heterochromatin-associated serpin that is an effective inhibitor of the papain-like cysteine proteases. Our laboratories have extensively investigated the dual functions of this protein, namely, chromatin condensation and protease inhibition. Unlike other serpins to date, MENT contains a unique insertion between the C- and D-helices known as the "M-loop." This loop contains two critical functional motifs that allow the nuclear function of MENT, namely, nuclear localization and DNA binding. However, the nuclear function of MENT is not restricted to the activities of the M-loop alone. In vitro, MENT brings about the dramatic remodeling of chromatin into higher-order structures by forming protein bridges via its reactive center loop. Further, we have determined that in a protease-mediated effect, DNA can act as a cofactor to accelerate the rate at which MENT can inhibit its target proteases. In this chapter, we discuss the isolation of MENT from native chicken blood as well as recombinant protein produced in Escherichia coli. Various techniques including in vitro functional assays and biophysical characterization are explained that can be used to elucidate the ability of the protein to interact with DNA and other deoxynucleoprotein complexes. In situ chromatin precipitation using natively purified MENT is also detailed.
authors.affiliation: Department of Biochemistry & Molecular Biology, Penn State University College of Medicine, Milton S. Hershey Medical Center, University Drive, Hershey, Pennsylvania, USA.
authors.forename: Sergei
authors.initials: S
authors.lastname: Grigoryev
authors.forename: Sheena
authors.initials: S
authors.lastname: McGowan
mesh_terms: D000818:Animals; D030161:Avian Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D002843:Chromatin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D004926:Escherichia coli; D017404:In Situ Hybridization, Fluorescence; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D015843:Serpins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D014169:Transformation, Bacterial
chemical_list: D030161:Avian Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; C081441:MENT protein, Gallus gallus; D009707:Nucleosomes; D011994:Recombinant Proteins; D015843:Serpins; D004247:DNA