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Proteome exploration to provide a resource for the investigation of Ganoderma lucidum.

Yu GJ, Yin YL, Yu WH, Liu W, Jin YX, Shrestha A, Yang Q, Ye XD, Sun H - PLoS ONE (2015)

Bottom Line: Among these proteins, 61 lignocellulose degrading proteins were detected, most of which (49 proteins) were found in the 90-day fruiting bodies.Based on the results from the proteomic and sequence alignment analyses, a potentially new immunomodulatory protein (GL18769) was expressed and shown to have high immunomodulatory activity.In this study, proteomic and biochemical analyses of G. lucidum were performed for the first time, revealing that proteins from this fungus can play significant bioactive roles and providing a new foundation for the further functional investigations that this fungus merits.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.

ABSTRACT
Ganoderma lucidum is a basidiomycete white rot fungus that has been used for medicinal purposes worldwide. Although information concerning its genome and transcriptome has recently been reported, relatively little information is available for G. lucidum at the proteomic level. In this study, protein fractions from G. lucidum at three developmental stages (16-day mycelia, and fruiting bodies at 60 and 90 days) were prepared and subjected to LC-MS/MS analysis. A search against the G. lucidum genome database identified 803 proteins. Among these proteins, 61 lignocellulose degrading proteins were detected, most of which (49 proteins) were found in the 90-day fruiting bodies. Fourteen TCA-cycle related proteins, 17 peptidases, two argonaute-like proteins, and two immunomodulatory proteins were also detected. A majority (470) of the 803 proteins had GO annotations and were classified into 36 GO terms, with "binding", "catalytic activity", and "hydrolase activity" having high percentages. Additionally, 357 out of the 803 proteins were assigned to at least one COG functional category and grouped into 22 COG classifications. Based on the results from the proteomic and sequence alignment analyses, a potentially new immunomodulatory protein (GL18769) was expressed and shown to have high immunomodulatory activity. In this study, proteomic and biochemical analyses of G. lucidum were performed for the first time, revealing that proteins from this fungus can play significant bioactive roles and providing a new foundation for the further functional investigations that this fungus merits.

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Related in: MedlinePlus

Protein preparation from three different developmental stages of G. lucidum.(A) Protein preparation of mycelium (16 days) and fruiting bodies at 60 days (60dF). The grids indicate how the SDS-PAGE gel bands (16dM1–16dM5, 60dF1–60dF5) were cut for MS identification. The middle lane represents the molecular weight of the markers (kDa). (B) Workflow of protein separation from fruiting bodies at 90 days (90dF). (C) SDS-PAGE of the DEAE column elution fractions of the 90dF total proteins. The gel was silver stained and prepared as two fractions (90dElu1 and 90dElu2) before mass spectrometry. (D) The DEAE flow through fraction of total proteins from 90dF (90dF1–90dF9) was separated by HPLC. The x-axis represents the run time of HPLC method, the left y-axis shows the absorbance value of proteins at 280 nm and the right y-axis indicates the acetonitrile concentration. (E) The 9 HPLC fractions were dialyzed, lyophilized and subjected to SDS-PAGE and silver staining.
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pone.0119439.g002: Protein preparation from three different developmental stages of G. lucidum.(A) Protein preparation of mycelium (16 days) and fruiting bodies at 60 days (60dF). The grids indicate how the SDS-PAGE gel bands (16dM1–16dM5, 60dF1–60dF5) were cut for MS identification. The middle lane represents the molecular weight of the markers (kDa). (B) Workflow of protein separation from fruiting bodies at 90 days (90dF). (C) SDS-PAGE of the DEAE column elution fractions of the 90dF total proteins. The gel was silver stained and prepared as two fractions (90dElu1 and 90dElu2) before mass spectrometry. (D) The DEAE flow through fraction of total proteins from 90dF (90dF1–90dF9) was separated by HPLC. The x-axis represents the run time of HPLC method, the left y-axis shows the absorbance value of proteins at 280 nm and the right y-axis indicates the acetonitrile concentration. (E) The 9 HPLC fractions were dialyzed, lyophilized and subjected to SDS-PAGE and silver staining.

Mentions: Approximately 100 g of the 90-day fruiting bodies (90dF) were crushed into a fine powder and extracted twice with 1.5 L cold 0.01 M PBS (pH 8.5) and 10 EDTA-free proteinase inhibitor cocktail tablets at 4°C for 24 hours. The supernatant was collected by centrifugation at 12,000 × g for 20 min at 4°C and loaded onto a DEAE Sepharose Fast Flow (GE Healthcare) column equilibrated with 10 mM PBS (Fig. 2B). The bound materials were eluted with the same buffer containing 1 M NaCl. Both the flow through fraction and the eluate were collected. The flow through fraction was further separated by reverse phase high-performance liquid chromatography (RP-HPLC) using an RP-HPLC column (Flexar, PerkinElmer, C18 column, 10 × 250 mm). The elution was carried out with a 0% to 30% gradient of acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA) at 2 ml/min for 35 min, and then with a 30% to 40% gradient of acetonitrile in 0.1% TFA at 0.8 ml/min for 85 min. Nine fractions were collected (Fig. 2D), and each was dialyzed extensively against distilled water and lyophilized. The DEAE column eluate (with 1 mM NaCl) and nine HPLC fractions were separately concentrated by the TCA/acetone method described above.


Proteome exploration to provide a resource for the investigation of Ganoderma lucidum.

Yu GJ, Yin YL, Yu WH, Liu W, Jin YX, Shrestha A, Yang Q, Ye XD, Sun H - PLoS ONE (2015)

Protein preparation from three different developmental stages of G. lucidum.(A) Protein preparation of mycelium (16 days) and fruiting bodies at 60 days (60dF). The grids indicate how the SDS-PAGE gel bands (16dM1–16dM5, 60dF1–60dF5) were cut for MS identification. The middle lane represents the molecular weight of the markers (kDa). (B) Workflow of protein separation from fruiting bodies at 90 days (90dF). (C) SDS-PAGE of the DEAE column elution fractions of the 90dF total proteins. The gel was silver stained and prepared as two fractions (90dElu1 and 90dElu2) before mass spectrometry. (D) The DEAE flow through fraction of total proteins from 90dF (90dF1–90dF9) was separated by HPLC. The x-axis represents the run time of HPLC method, the left y-axis shows the absorbance value of proteins at 280 nm and the right y-axis indicates the acetonitrile concentration. (E) The 9 HPLC fractions were dialyzed, lyophilized and subjected to SDS-PAGE and silver staining.
© Copyright Policy
Related In: Results  -  Collection

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pone.0119439.g002: Protein preparation from three different developmental stages of G. lucidum.(A) Protein preparation of mycelium (16 days) and fruiting bodies at 60 days (60dF). The grids indicate how the SDS-PAGE gel bands (16dM1–16dM5, 60dF1–60dF5) were cut for MS identification. The middle lane represents the molecular weight of the markers (kDa). (B) Workflow of protein separation from fruiting bodies at 90 days (90dF). (C) SDS-PAGE of the DEAE column elution fractions of the 90dF total proteins. The gel was silver stained and prepared as two fractions (90dElu1 and 90dElu2) before mass spectrometry. (D) The DEAE flow through fraction of total proteins from 90dF (90dF1–90dF9) was separated by HPLC. The x-axis represents the run time of HPLC method, the left y-axis shows the absorbance value of proteins at 280 nm and the right y-axis indicates the acetonitrile concentration. (E) The 9 HPLC fractions were dialyzed, lyophilized and subjected to SDS-PAGE and silver staining.
Mentions: Approximately 100 g of the 90-day fruiting bodies (90dF) were crushed into a fine powder and extracted twice with 1.5 L cold 0.01 M PBS (pH 8.5) and 10 EDTA-free proteinase inhibitor cocktail tablets at 4°C for 24 hours. The supernatant was collected by centrifugation at 12,000 × g for 20 min at 4°C and loaded onto a DEAE Sepharose Fast Flow (GE Healthcare) column equilibrated with 10 mM PBS (Fig. 2B). The bound materials were eluted with the same buffer containing 1 M NaCl. Both the flow through fraction and the eluate were collected. The flow through fraction was further separated by reverse phase high-performance liquid chromatography (RP-HPLC) using an RP-HPLC column (Flexar, PerkinElmer, C18 column, 10 × 250 mm). The elution was carried out with a 0% to 30% gradient of acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA) at 2 ml/min for 35 min, and then with a 30% to 40% gradient of acetonitrile in 0.1% TFA at 0.8 ml/min for 85 min. Nine fractions were collected (Fig. 2D), and each was dialyzed extensively against distilled water and lyophilized. The DEAE column eluate (with 1 mM NaCl) and nine HPLC fractions were separately concentrated by the TCA/acetone method described above.

Bottom Line: Among these proteins, 61 lignocellulose degrading proteins were detected, most of which (49 proteins) were found in the 90-day fruiting bodies.Based on the results from the proteomic and sequence alignment analyses, a potentially new immunomodulatory protein (GL18769) was expressed and shown to have high immunomodulatory activity.In this study, proteomic and biochemical analyses of G. lucidum were performed for the first time, revealing that proteins from this fungus can play significant bioactive roles and providing a new foundation for the further functional investigations that this fungus merits.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.

ABSTRACT
Ganoderma lucidum is a basidiomycete white rot fungus that has been used for medicinal purposes worldwide. Although information concerning its genome and transcriptome has recently been reported, relatively little information is available for G. lucidum at the proteomic level. In this study, protein fractions from G. lucidum at three developmental stages (16-day mycelia, and fruiting bodies at 60 and 90 days) were prepared and subjected to LC-MS/MS analysis. A search against the G. lucidum genome database identified 803 proteins. Among these proteins, 61 lignocellulose degrading proteins were detected, most of which (49 proteins) were found in the 90-day fruiting bodies. Fourteen TCA-cycle related proteins, 17 peptidases, two argonaute-like proteins, and two immunomodulatory proteins were also detected. A majority (470) of the 803 proteins had GO annotations and were classified into 36 GO terms, with "binding", "catalytic activity", and "hydrolase activity" having high percentages. Additionally, 357 out of the 803 proteins were assigned to at least one COG functional category and grouped into 22 COG classifications. Based on the results from the proteomic and sequence alignment analyses, a potentially new immunomodulatory protein (GL18769) was expressed and shown to have high immunomodulatory activity. In this study, proteomic and biochemical analyses of G. lucidum were performed for the first time, revealing that proteins from this fungus can play significant bioactive roles and providing a new foundation for the further functional investigations that this fungus merits.

Show MeSH
Related in: MedlinePlus