Document 0473
 DOCN  M9490473
 TI    Nucleolar protein B23: bacterial expression, purification,
       oligomerization and secondary structures of two isoforms.
 DT    9411
 AU    Umekawa H; Chang JH; Correia JJ; Wang D; Wingfield PT; Olson MO;
       Department of Biochemistry, University of Mississippi Medical; Center,
       Jackson 39216-4505.
 SO    Cell Mol Biol Res. 1993;39(7):635-45. Unique Identifier : AIDSLINE
       MED/94332167
 AB    Protein B23 is an abundant nucleolar phosphoprotein and putative
       ribosome assembly factor. Two forms of the protein, B23.1 and B23.2,
       contain 292 and 257 amino acids, respectively, and differ only in their
       C-terminal sequences. The two B23 isoforms have been produced in
       Escherichia coli using the pKK223-3 expression vector and purified to
       near homogeneity. The purification utilized ammonium sulfate
       fractionation followed by chromatography on DEAE-cellulose,
       heparin-Sepharose and Bio-Rad Q. By combined gel filtration and
       sedimentation analyses, both B23.1 and B23.2 formed multimers of Mr 210
       to 255 kDa (apparent hexamers), suggesting that the differences in
       C-terminal ends of of the isoforms do not affect oligomerization. The
       oligomerization was not dependent on disulfide bond formation. The
       circular dichroism spectra of recombinant proteins B23.1 and B23.2 were
       similar suggesting that the carboxyl-terminal difference in the two
       proteins does not markedly influence overall secondary structure. Using
       routines for fitting the CD spectra to those of basis vectors the
       recombinant B23 isoforms appeared to be composed predominantly of
       beta-sheet and beta-turn secondary structures. Protein B23 from HeLa
       cell nuclei was recently shown to have a high affinity for the HIV-1 Rev
       protein. Using sucrose density gradient centrifugation it was shown that
       both recombinant proteins B23.1 and B23.2, as well as B23.1 isolated
       from Novikoff hepatoma nucleoli, were capable of binding the Rev
       protein.
 DE    Cell Nucleus/METABOLISM  Centrifugation, Density Gradient
       Chromatography, Affinity  Chromatography, DEAE-Cellulose
       Chromatography, Gel  Chromatography, Ion Exchange  Circular Dichroism
       Cloning, Molecular/METHODS  Escherichia coli/*METABOLISM  Gene Products,
       rev/METABOLISM  Hela Cells  Human  HIV-1/METABOLISM  Macromolecular
       Systems  Nuclear Proteins/*BIOSYNTHESIS/*CHEMISTRY/ISOLATION & PURIF
       Phosphoproteins/BIOSYNTHESIS/CHEMISTRY  Plasmids  *Protein Structure,
       Secondary  Recombinant Proteins/*BIOSYNTHESIS/CHEMISTRY/ISOLATION &
       PURIF  Restriction Mapping  Support, U.S. Gov't, P.H.S.  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).