We have set up methods for intracellular and extracellular proteome analysis in Trichoderma. These include analysis of steady state total protein pattern using 2D gel electrophoresis as well as detection of actively synthesised proteins using metabolic labelling. In addition, phosphoprotein staining and Western analysis with phosphoprotein antibodies have been used to detect differences in the cellular levels of phosphorylated proteins. Proteome analysis has been applied to study the cellular responses activated in Trichoderma reesei upon production of a heterologous protein, tPA (tissue plasminogen activator). Protein samples from chemostat cultures of the tPA-producing transformant and the parental strain Rut-C30 were subjected to 2D gel electrophoresis and protein spots with altered intensity were identified using LC-MS/MS analysis and comparison of the obtained peptide masses and sequence tags with public protein sequence data bases. The analysis revealed up-regulation of a number of proteins involved in protein glycosylation and folding, and members of heat shock protein families. Many of these proteins have not been previously reported from T. reesei. The responses detected in the strain producing the heterologous protein were also compared to those observed in cultures treated with chemical agents to inhibit protein folding and transport, dithiothreitol (DTT) and Brefeldin A, respectively.
|Publication status||Published - 2004|
|MoE publication type||Not Eligible|
|Event||7th European Conference on Fungal Genetics - Copenhagen, Denmark|
Duration: 17 Apr 2004 → 20 Apr 2004
|Conference||7th European Conference on Fungal Genetics|
|Period||17/04/04 → 20/04/04|