Comparison of lysis methods and preparation protocols for one‐ and two‐dimensional electrophoresis of Aspergillus oryzae intracellular proteins

Filamentous fungal fermentations are used to produce billions of dollars of biochemical and pharmaceutical products annually, yet are plagued by a number of poorly understood problems that would benefit from proteomic analysis. Unfortunately, few publications are available which describe extraction of filamentous fungal proteins for two‐dimensional electrophoresis. The goal here was to develop protocols for extraction of fungal proteins, from both wild‐type and a recombinant strain of the industrially important filamentous fungi Aspergillus oryzae , to be used for both one‐ and two‐dimensional electrophoresis (1‐DE and 2‐DE). Because fungal cell walls are exceptionally resistant to fragmentation, four lysis protocols were tested: (i) boiling in strong alkali solution, (ii) boiling in Sodium dodecyl surfate (SDS), (iii) chemical lysis in Y‐PER® reagent, and (iv) mechanical lysis via rapid agitation with glass beads in a Mini‐BeadBeater®. For both 1‐DE and 2‐DE, rapid agitation with glass beads was found to be the most efficient extraction method, yielding both mini‐ and large‐format gels with little streaking or spot tailing, and proteins comprising a broad range of molecular weights and p I values.