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

✍ Scribed by M. P. Nandakumar; Mark R. Marten

• Publisher: John Wiley and Sons
• Year: 2002
• Tongue: English
• Weight: 340 KB
• Volume: 23
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/1522-2683(200207)23:14<2216::aid-elps2216>3.0.co;2-y

✦ Synopsis

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(R) reagent, and (iv) mechanical lysis via rapid agitation with glass beads in a Mini-BeadBeater(R). 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 pI values.