In the Methods, we describe the comprehensive protocol used to obtain the soluble protein extracts. Briefly, to improve cell disruption and minimize proteolysis, lyophilized yeast cells were vortexed directly with glass beads. Lysis buffer and protease inhibitors were then added to reduce proteolytic enzyme activity. The pellet was disrupted MEK162 five times in a RiboLyzer, followed by phenol extraction and methanol precipitation. Finally, the protein spots were stained with Coomassie and identified by MALDI-TOF MS. To obtain the protein profiles of X. dendrorhous, the yeast was cultured in MM-glucose and harvested at the lag, late exponential and stationary growth phases.
Four independent cultures showed continuous increases in cell density until 70 h, which was immediately prior to the induction of carotenoid biosynthesis (Figure 1). As we have previously reported, pigment accumulation in MM-glucose was evident Selleck VS-4718 during the stationary phase [22, 23]. Carotenoid analysis by HPLC showed that astaxanthin was the main
carotenoid (75-90% of the total carotenoids) produced by the yeast during growth. Figure 1 Growth and pigment production in X. dendrorhous. Growth was measured by the absorbance at 560 nm (shown CP673451 clinical trial on a log scale), which is represented by the squares and solid line. The means ± SD of the values obtained from four independent cultures are shown. The vertical arrows indicate the harvest times for the assays (24, 70 and 96 h, which corresponded
to lag, late Loperamide exponential and stationary growth phases, respectively). The solid line represents the total carotenoids. The asterisk indicates the induction of carotenoid biosynthesis. For the proteomic analyses, triplicate protein extracts (prepared from three independent cultures) were subjected to 2D analysis, and their protein profiles were obtained. The different protein profiles were subjected to a stringent comparative analysis using PDQuest software (version 7.1.1, Bio-Rad). After automated spot detection, spots were checked manually to eliminate possible artifacts such as background noise or streaks. Student’s t-test (p < 0.05) was used to determine whether the relative changes in protein abundance were statistically significant. A representative 2D image is shown in Figure 2. The protein data analyses showed a consistent protein profile during growth (See additional file 1, Fig. S1). On average, approximately 600 spots were detected on each 2D gel in a pI-range of 3-10 and a molecular mass range of 10-100 kDa. This pattern of proteins was highly reproducible, and similar results were obtained in the triplicate cell extracts. Overall, the protein profiles did not change dramatically (over 90% of the spots were identical) during growth. Of the spots detected in all gels, 450 spots with different intensities were selected to be excised, digested with trypsin and analyzed by MALDI-TOF MS for protein identification.