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

Abstract

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.

Proteome analysis of Aspergillus ochraceus

“…Cell lysis via mechanical agitation with glass beads was performed as previously described (Nandakumar and Marten 2002) with slight modifications. Briefly, harvested mycelia were washed with 300 ml each of PBS (0.9% w/v NaCl in 0.1 M phosphate buffer, pH 7.0) and water.…”

“…Filamentous fungi have an exceptionally strong cell wall and thus, in sample preparation for electrophoresis, cell lysis is often the most difficult step. Several researchers (Melin et al 2002;Nandakumar et al 2003;Grinyer et al 2004;Strom et al 2005) used mechanical lysis via glass beads to liberate cytoplasmic proteins, and this approach has been reported to be more efficient than either chemical or enzymatic extraction methods (Nandakumar and Marten 2002). However, the most widely used method for extraction seems to be grinding in liquid nitrogen using mortar and pestle (Hernandez-Macedo et al 2002;Grinyer et al 2005;Shimizu and Wariishi 2005;Kniemeyer et al 2006;Yajima and Kav 2006).…”

Crucial Role of Antioxidant Proteins and Hydrolytic Enzymes in Pathogenicity of Penicillium expansum

“…Mycelia were collected after incubation for 48, 60, and 72 h and washed thoroughly with 10 mM potassium phosphate buffer, pH 7.0, to remove bound extracellular proteins and other contaminants. The intercellular proteins were extracted following the method of Kim et al (27) and Nandakumar and Marten (23). Mycelia were lysed by sonification (with cooling on ice) with 1 ml of lysis buffer containing 0.5 M Tris-HCl, pH 8.3, 2% (v/v) Nonidet P-40, 20 mM MgCl 2 , 2% (v/v) ␤-mercaptoethanol, and 1 mM PMSF.…”

“…In recent years, proteomics analysis has proven to be a powerful method for studying the changes of protein expression profiles in response to various stresses in yeast (16 -18) and bacterium (19 -22). On the other hand, there is still a lack of information about proteomics analysis from filamentous fungi (23,24). P. expansum is very poorly characterized at the protein level.…”

Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: Identification and characterisation of an elongation factor 1B protein with glutathione transferase activity

“…Many Aspergillus species have merited attention from a biotechnological standpoint, as a consequence of a secreted proteome rich in industrially useful enzymes [4]. Until recently, however, little information was available with respect to either the technical requirements for protein extraction or the proteome content of most Aspergillus species.…”