NUCLEOLAR PROTEIN B23 - BACTERIAL EXPRESSION, PURIFICATION, OLIGOMERIZATION AND SECONDARY STRUCTURES OF 2 ISOFORMS

Protein B23 is an abundant nucleolar phosphoprotein and putative ribos ome assembly factor. Two forms of the protein, B23.1 and B23.2, contai n 292 and 257 amino acids, respectively, and differ only in their C-te rminal sequences. The two B23 isoforms have been Produced in Escherich ia coli using the pKK223-3 expression vector and purified to near homo geneity. The purification utilized ammonium sulfate fractionation foll owed by chromatography on DEAE-cellulose, heparin-Sepharose and Bio-Ra d 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 the isoforms do not affect oligomerization. The oligomerization was not dependent on d isulfide bond formation. The circular dichroism spectra of recombinant proteins B23.1 and B23.2 were similar suggesting that the carboxyl-te rminal difference in the two proteins does not markedly influence over all secondary structure. Using routines for fitting the CD spectra to those of basis vectors the recombinant B23 isoforms appeared to be com posed predominantly of beta-sheet and beta-turn secondary structures. Protein B23 from HeLa cell nuclei was recently shown to have a high af finity for the HIV-1 Rev protein. Using sucrose density gradient centr ifugation it was shown that both recombinant proteins B23:1 and B23.2, as well as B23.1 isolated from Novikoff hepatoma nucleoli, were capab le of binding the Rev protein.