Figure 2

SDS-PAGE of recombinant MuHN and MeH protein fractions insoluble in 8 M urea solution. (A-D) The same samples were run on each gel: control sample from S. cerevisiae cells, transformed with empty vector was loaded on lane 1, whereas samples from S. cerevisiae expressing recombinant viral proteins were loaded on lanes 2 (MuHN) and 3 (MeH), respectively. (A) Coomassie blue-stained gel. Long solid arrows indicate major, whereas dotted arrows - minor forms of partially purified recombinant MuHN (lane 2) and MeH (lane 3). Short arrow points to ~50 kDa band analysed by MS directly from 1-D SDS-PAGE gel (the band number 15 is given according to the list of identified yeast proteins in Table 1). (B) Western blot using monoclonal antibody 782 to the native MuHN. In addition to the main band of ~60 kDa (solid arrow) the minor band (up to 65 kDa, dotted arrow) of MuHN was also distinguished (lane 2). Mab 782 cross-reacted with both major and minor bands of recombinant MeH (lane 3). (C) Western blot using anti-His antibody. Both ~65 kDa and ~75 kDa forms of MeH protein (indicated by solid and dotted arrows, respectively) were insoluble under mild denaturing conditions in 8 M urea solution (lane 3). (D) Western blot using Concanavalin A. The major forms of recombinant MuHN (~60 kDa) and MeH (~65 kDa) appeared to be non-glycosylated as they did not react with Concanavalin A (white band areas below dotted arrows in lanes 2 and 3, respectively). Only heterogeneous band of minor MeH form (~75 kDa) contained N-glycosylated protein reacting with Concanavalin A (lane 3, dotted arrow), similar result was observed in the case of MuHN minor form (lane 2).