Complex formation of purified p34.

(A) p34 was expressed in Escherichia coli and purified using conventional methods. Lane 1, inclusion bodies collected by centrifugation; lane 2, proteins dissolved in presence of 8 M urea and precipitated by ammonium sulfate (30% saturation); lane 3, eluate from Phenyl-Sepharose; lane 4, flow through from DEAE-Sephacel column. The proteins were separated by SDS-PAGE and the gel was stained by Coomassie. (B) CD spectra of p34. The purified protein was dissolved in 8 mM N-Decyl-β-D-Maltopyranosid, 10 mM KCl, 20 mM K₂HPO₄/KH₂PO₄, pH 7.0 and analyzed using a Jasco J-810 spectrapolarimeter. (C) Chemical cross-linking. Purified p34 was incubated with 50 µM DSS (lane 2) or 0.5 mM Sulfo-MBS (lanes 3 and 4) for 30 min at 0°C. The reaction was stopped by addition of 0.5 M Tris-HCl pH 7.4, the proteins were precipitated by TCA, separated by SDS-PAGE, transferred on nitrocellulose, and labelled using a polyclonal antiserum. (D) Analysis of p34 in BN-PAGE. Purified p34 was dissolved in 0.5% Triton X-100, 10% Glycerol, 50 mM NaCl, 0.1 mM EDTA, PMSF 1 mM, 20 mM Tris-HCl pH 7.0, and applied on a gel for BN-PAGE in the presence of 500 mM ε-aminocaproic acid (first dimension). A lane from the gel was excised and layered on top of a conventional SDS-PAGE for separation of the proteins under denaturing conditions (second dimension). The proteins were transferred on nitrocellulose and labelled using a polyclonal antiserum directed against p34 (upper panel). Truncated p34 comprising residues 37–319 was similarly analyzed (middle panel), or dissolved in the presence of 8 M urea and 1% dodecylmaltoside for separation in the first dimension (lower panel).