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Experimental and computational analysis of the secretome of the hyperthermophilic archaeon Pyrococcus furiosus.

Schmid G, Mathiesen G, Arntzen MO, Eijsink VG, Thomm M - Extremophiles (2013)

Bottom Line: Most of the 43 proteins without predicted N-terminal signal sequences are known to have intracellular functions, mainly (70 %) related to intracellular metabolism.In silico analyses indicated that the genome of P. furiosus encodes 145 proteins with N-terminal SPs, including 21 putative lipoproteins and 17 with a class III peptide.The putative lipoprotein signal peptides have a unique sequence motif, distinct from the motifs in bacteria and other archaeal orders.

View Article: PubMed Central - PubMed

Affiliation: Hyperthermics Regensburg GmbH, Josef-Engert-Straße 9, 93053, Regensburg, Germany.

ABSTRACT
Although Pyrococcus furiosus is one of the best studied hyperthermophilic archaea, to date no experimental investigation of the extent of protein secretion has been performed. We describe experimental verification of the extracellular proteome of P. furiosus grown on starch. LC-MS/MS-based analysis of culture supernatants led to the identification of 58 proteins. Fifteen of these proteins had a putative N-terminal signal peptide (SP), tagging the proteins for translocation across the membrane. The detected proteins with predicted SPs and known function were almost exclusively involved in important extracellular functions, like substrate degradation or transport. Most of the 43 proteins without predicted N-terminal signal sequences are known to have intracellular functions, mainly (70 %) related to intracellular metabolism. In silico analyses indicated that the genome of P. furiosus encodes 145 proteins with N-terminal SPs, including 21 putative lipoproteins and 17 with a class III peptide. From these we identified 15 (10 %; 7 SPI, 3 SPIII and 5 lipoproteins) under the specific growth conditions of this study. The putative lipoprotein signal peptides have a unique sequence motif, distinct from the motifs in bacteria and other archaeal orders.

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Related in: MedlinePlus

A representative coomassie stained SDS-PAGE gel showing concentrated proteins from P. furiosus cell-free culture supernatant. 1 PageRuler Prestained Protein Ladder; molecular weights are indicated in kDa. 2 37 μg protein from culture supernatant
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Fig1: A representative coomassie stained SDS-PAGE gel showing concentrated proteins from P. furiosus cell-free culture supernatant. 1 PageRuler Prestained Protein Ladder; molecular weights are indicated in kDa. 2 37 μg protein from culture supernatant

Mentions: Supernatant fractions from P. furiosus grown on starch as major carbon source were collected in the late exponential growth phase, using anaerobic conditions during harvesting to prevent cell lysis. The proteins in the cell-free supernatant were concentrated by ultrafiltration followed by acetone precipitation. The concentrated proteins were separated by 1D-gel electrophoresis (Fig. 1) and converted to tryptic peptides by in-gel trypsination. The peptides were then analyzed using high resolution LC–MS/MS as described in the “Materials and methods” section.Fig. 1


Experimental and computational analysis of the secretome of the hyperthermophilic archaeon Pyrococcus furiosus.

Schmid G, Mathiesen G, Arntzen MO, Eijsink VG, Thomm M - Extremophiles (2013)

A representative coomassie stained SDS-PAGE gel showing concentrated proteins from P. furiosus cell-free culture supernatant. 1 PageRuler Prestained Protein Ladder; molecular weights are indicated in kDa. 2 37 μg protein from culture supernatant
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3824201&req=5

Fig1: A representative coomassie stained SDS-PAGE gel showing concentrated proteins from P. furiosus cell-free culture supernatant. 1 PageRuler Prestained Protein Ladder; molecular weights are indicated in kDa. 2 37 μg protein from culture supernatant
Mentions: Supernatant fractions from P. furiosus grown on starch as major carbon source were collected in the late exponential growth phase, using anaerobic conditions during harvesting to prevent cell lysis. The proteins in the cell-free supernatant were concentrated by ultrafiltration followed by acetone precipitation. The concentrated proteins were separated by 1D-gel electrophoresis (Fig. 1) and converted to tryptic peptides by in-gel trypsination. The peptides were then analyzed using high resolution LC–MS/MS as described in the “Materials and methods” section.Fig. 1

Bottom Line: Most of the 43 proteins without predicted N-terminal signal sequences are known to have intracellular functions, mainly (70 %) related to intracellular metabolism.In silico analyses indicated that the genome of P. furiosus encodes 145 proteins with N-terminal SPs, including 21 putative lipoproteins and 17 with a class III peptide.The putative lipoprotein signal peptides have a unique sequence motif, distinct from the motifs in bacteria and other archaeal orders.

View Article: PubMed Central - PubMed

Affiliation: Hyperthermics Regensburg GmbH, Josef-Engert-Straße 9, 93053, Regensburg, Germany.

ABSTRACT
Although Pyrococcus furiosus is one of the best studied hyperthermophilic archaea, to date no experimental investigation of the extent of protein secretion has been performed. We describe experimental verification of the extracellular proteome of P. furiosus grown on starch. LC-MS/MS-based analysis of culture supernatants led to the identification of 58 proteins. Fifteen of these proteins had a putative N-terminal signal peptide (SP), tagging the proteins for translocation across the membrane. The detected proteins with predicted SPs and known function were almost exclusively involved in important extracellular functions, like substrate degradation or transport. Most of the 43 proteins without predicted N-terminal signal sequences are known to have intracellular functions, mainly (70 %) related to intracellular metabolism. In silico analyses indicated that the genome of P. furiosus encodes 145 proteins with N-terminal SPs, including 21 putative lipoproteins and 17 with a class III peptide. From these we identified 15 (10 %; 7 SPI, 3 SPIII and 5 lipoproteins) under the specific growth conditions of this study. The putative lipoprotein signal peptides have a unique sequence motif, distinct from the motifs in bacteria and other archaeal orders.

Show MeSH
Related in: MedlinePlus