Results and Discussion
Sup35 was consistently eluted in a stepwise manner. Two peaks were
observed from non-denatured Ni2+ affinity
chromatography (Fig 1A). Peak I was dissociated into three main bands on
SDS-PAGE (Fig 1B), the Sup35 monomer band, an undetermined band
(approximately 45 kDa, named as X1), and an outer membrane protein band
(Omp F) [26]. The assignment of Omp F is based on its molecular
weight shown on SDS-PAGE, the intensity of the band and the mass
spectrum result (Table S1). The dissociation of peak I on SDS-PAGE had
many continuous bands between the monomer and X1, which were defined as
zone X2 (Fig 1B). Peak II exhibited a monomer band on SDS-PAGE with an
intensity less than that under DTT. This indicates that part of the
species in peak II is aggregated (S-aggregate) and caused by inter-Sup35
disulfide bonds. No intensity change could be observed for the protein
bands associated with peak I on SDS-PAGE with the addition of DTT, thus
excluding the formation of inter-Sup35 disulfide bonds.