(334c) Pressure Enhanced Processes (PrEP) Enabling High Quality Two-Dimensional Gel Electrophoresis of Frankia Mycelia and Vesicle Structures

The actinobacteria, Frankia, are particularly challenging in terms of sample preparation, isoelectric focusing (IEF) and two-dimensional gel electrophoresis (2DE). While French Press is typically used for the disruption of Frankia mycelia, protein yields are typically very low and potentially biased toward cytoplasmic proteins. Operating at a maximum pressure of 20,000 psi, the French press fails to disrupt vesicles such that this recalcitrant organelle has remained largely uncharacterized in terms of its protein constituency. Alternatively, pressure cycling technology (PCT) recycles the sample at 45,000 psi maximum pressure and effectively disrupts both mycelia and the resilient vesicles. However, increased protein yield from Frankia strain EAN1pec corresponds to increased contamination by an indigenous red pigment which interferes with protein assay and IEF. The pigment is lessened, but not removed by acetone precipitation and concentrates with proteins in ultrafiltrative retentates, suggesting the pigment exists as high molecular weight aggregates which are not dissociated by chaotropes or detergents. If not removed, the pigment precipitates on the surface of the immobilized pH gradient (IPG) and prevents proteins from being imbibed into the strip. This is evidenced by the preponderance of horizontal streaking in second dimension gels. Pigment that is imbibed in the IPG rapidly focuses in the pH 4-5 region of the gradient, creating a highly conductive zone where a localized voltage drop prevents proteins from properly focusing. Hence, it is ambiguous as to whether proteins that are aligned with this zone are due to their association with the pigment or as an electrophoretic phenomenon. Ultrafiltration of mycelia and vesicle homogenates with porous membranes of 100 kDa nominal molecular weight limit partitioned 80% of the initial sample volume containing approximately 60% of the protein mass into the filtrate, whereas most of the offending pigment was accumulated in the retentate. Consequently, over 500 proteins were resolved in two-dimensional gels of vesicle filtrates.