Proteome analysis in Trichoderma reesei

Tiina Pakula, Tero Riipi, Heini Koivistoinen, Karin Lanthaler, Jaana Uusitalo, Markku Saloheimo, Leena Valmu, Nisse Kalkkinen, Geoff Robson, Merja Penttilä

Research output: Contribution to conferenceConference articleScientific

Abstract

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.
Original languageEnglish
Publication statusPublished - 2004
EventPhysiology of Yeasts and Filamentous Fungi (PYFF2): 121th Event of the European Federation of Biotechnology - Anglet, France
Duration: 24 Mar 200428 Mar 2004

Conference

ConferencePhysiology of Yeasts and Filamentous Fungi (PYFF2)
CountryFrance
CityAnglet
Period24/03/0428/03/04

Fingerprint

Trichoderma reesei
proteome
proteins
t-plasminogen activator
phosphoproteins
gel electrophoresis
brefeldin A
protein folding
protein transport
dithiothreitol
Trichoderma
glycosylation
heat shock proteins
amino acid sequences
peptides
antibodies

Keywords

  • 2D gel electrophoresis
  • protein production
  • heterologous protein
  • secretion stress

Cite this

Pakula, T., Riipi, T., Koivistoinen, H., Lanthaler, K., Uusitalo, J., Saloheimo, M., ... Penttilä, M. (2004). Proteome analysis in Trichoderma reesei. Paper presented at Physiology of Yeasts and Filamentous Fungi (PYFF2), Anglet, France.
Pakula, Tiina ; Riipi, Tero ; Koivistoinen, Heini ; Lanthaler, Karin ; Uusitalo, Jaana ; Saloheimo, Markku ; Valmu, Leena ; Kalkkinen, Nisse ; Robson, Geoff ; Penttilä, Merja. / Proteome analysis in Trichoderma reesei. Paper presented at Physiology of Yeasts and Filamentous Fungi (PYFF2), Anglet, France.
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abstract = "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.",
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Pakula, T, Riipi, T, Koivistoinen, H, Lanthaler, K, Uusitalo, J, Saloheimo, M, Valmu, L, Kalkkinen, N, Robson, G & Penttilä, M 2004, 'Proteome analysis in Trichoderma reesei' Paper presented at Physiology of Yeasts and Filamentous Fungi (PYFF2), Anglet, France, 24/03/04 - 28/03/04, .

Proteome analysis in Trichoderma reesei. / Pakula, Tiina; Riipi, Tero; Koivistoinen, Heini; Lanthaler, Karin; Uusitalo, Jaana; Saloheimo, Markku; Valmu, Leena; Kalkkinen, Nisse; Robson, Geoff; Penttilä, Merja.

2004. Paper presented at Physiology of Yeasts and Filamentous Fungi (PYFF2), Anglet, France.

Research output: Contribution to conferenceConference articleScientific

TY - CONF

T1 - Proteome analysis in Trichoderma reesei

AU - Pakula, Tiina

AU - Riipi, Tero

AU - Koivistoinen, Heini

AU - Lanthaler, Karin

AU - Uusitalo, Jaana

AU - Saloheimo, Markku

AU - Valmu, Leena

AU - Kalkkinen, Nisse

AU - Robson, Geoff

AU - Penttilä, Merja

N1 - CA2: BEL2 BEL1 CA: BEL

PY - 2004

Y1 - 2004

N2 - 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.

AB - 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.

KW - 2D gel electrophoresis

KW - protein production

KW - heterologous protein

KW - secretion stress

M3 - Conference article

ER -

Pakula T, Riipi T, Koivistoinen H, Lanthaler K, Uusitalo J, Saloheimo M et al. Proteome analysis in Trichoderma reesei. 2004. Paper presented at Physiology of Yeasts and Filamentous Fungi (PYFF2), Anglet, France.