Synthesis of recombinant MuHN and MeH causes overexpression of some cellular proteins in yeast. (A-C) SDS-PAGE of whole cell lysates on a 10% polyacrylamide gel. All lysates were prepared from galactose-induced yeast cells of S. cerevisiae pep4 strain, transformed with empty vector (lane 1) or plasmids expressing MuHN (native sequence - lane 2 and with His6-tag - lane 3) or His6-tagged MeH (lane 4). Lane M - prestained protein ladder. (A) Coomassie blue-stained gel. Solid arrows indicate bands of recombinant MuHN (lanes 2 and 3) and MeH (lane 4) proteins. Dashed arrow points to ~70 kDa main band of yeast cellular proteins overexpressed in response to synthesis of MuHN and MeH (lanes 2-4). (B) Western blot using anti-His antibody. Expression of His-tagged recombinant MeH protein was confirmed by immunoblot analysis that revealed two main forms: major band of ~65 kDa and upper double band of ~75 kDa (lane 4, there are also a faint band just above 65 kDa and some degradation products visible). His-tagged recombinant MuHN protein did not react with anti-His antibody (lane 3). (C) Western blot using monoclonal antibody 782 to the native MuHN. Both native and His-tagged MuHN sequence variants were detected as ~60 kDa bands (lanes 2 and 3, respectively) corresponding to those shown in coomassie stained gel. Due to stronger reaction of Mab 782 with the native sequence MuHN variant the latter was chosen for further MuHN expression study. There was also some non-specific reaction and cross-reactivity with MeH observed.