Nucleolar protein B23: bacterial expression, purification, oligomerization and secondary structures of two isoforms.
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.
Umekawa
H
H
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505.
Chang
J H
JH
Correia
J J
JJ
Wang
D
D
Wingfield
P T
PT
Olson
M O
MO
D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism*; D015694:Gene Products, rev / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D000090243:Nucleophosmin; D010750:Phosphoproteins / Q000096:biosynthesis / Q000737:chemistry; D010957:Plasmids; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D054321:rev Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D000090243:Nucleophosmin