Figure 1.
Localization of Pb MLSr. (A) SDS-PAGE analysis of PbMLSr. E. coli BL21 C41 cells harboring the pET-32a-MLS plasmid were grown at 37°C to an OD600 of 0.6 and harvested before (lane 1) and after induction with 1 mM IPTG (lane 2). The cells were lysed by sonication, and the recombinant His-, Trx-, and S-Tagged PbMLS were isolated by affinity chromatography (lane 3). Tags were removed by EKMax™ Enterokinase digestion (lane 4). (B) Western blots of fusion PbMLSr (lane 1), cleaved PbMLSr (lane 2), crude extract proteins from yeast cells (lane 3), SDS-extracted yeast cell wall proteins (lane 4), and yeast cell wall proteins (lane 5). Proteins were probed with anti-PbMLSr antibody or with pre-immune rabbit (C). (D) Western blots of proteins of culture filtrate of P. brasiliensis yeast cells harvested after 24 h (lane 1), 36 h (lane 2), 7 days (lane 3), and 14 days (lane 4) of culture, and culture filtrate without P. brasiliensis as negative control (lane 5). Proteins were probed with anti-PbMLSr antibody or with pre-immune rabbit (E). (F) Western blots of peroxisomal fraction (lane 1) and mitochondrial fraction (lane 2) proteins from P. brasiliensis yeast cells were probed with anti-PbMLSr antibody. Molecular mass markers are indicated at the side.