Šárka cepáková, sandra stachová leader: mgr. markéta foldynová 28. 07. 2011 protein...
TRANSCRIPT
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Šárka Cepáková, Sandra Stachová
Leader: Mgr. Markéta Foldynová
28. 07. 2011
Protein glycosylations in cyanobacterium Synechocystis
Schola ludus
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Introduction
• Subject of research - post-translational modification - glycosylation
• Glycosylation: attachment of glycans to proteins (N-linked glycans, O-linked glycans)
• Model organism – cyanobacterium Synechocystis sp. PCC 6803
• Differences in cultivation: in the presence of glucose X autotrophic cultivation
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Hypotheses and aims
How does glucose affect cultivation of cyanobacteria Synechocystis?
• Effect on the biosynthesis of proteins and pigments
• Reason: alternative glycosylation of an enzyme or regulatory factor
• Expectation: different glycosylation pattern in some proteins after cultivation in presence of glucose
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Methods IIsolation of glycoproteins from cell extract: Glycoprotein Isolation Kit, ConA
Fig.1: Purification column
Separation: SDS-PAGE electrophoresis
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Methods II
Gel staining:
• Coomassie Blue Staining• Pierce Glycoprotein Staining KitPhotographs taken by sensitive CCD camera
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Results
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a) Comparison glucose cultivation X autotrophic cultivation
Cell membrane fraction I
Fig.2: Autotrophic cultivation (SDS-PAGE, coomassie) Fig.3: Glucose cultivation (SDS-PAGE, coomassie)
kDa250
130
95
72
55
36
2817
10
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b) Comparison glucose cultivation X autotrophic cultivation
Cell membrane fraction II
Fig.4: Autotrophic cultivation (SDS-PAGE, coomassie) Fig.5: Glucose cultivation (SDS-PAGE, coomassie)
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c) Comparison glucose cultivation X autotrophic cultivation
Soluble proteins
Fig.6: Autotrophic cultivation (SDS-PAGE, coomassie) Fig.7: Glucose cultivation (SDS-PAGE, coomassie)
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d) Comparison glucose cultivation X autotrophic cultivation
Y. H. Kim et al. (2009) Proteomics
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Conclusions
• Some of N-glycosylated proteins are unique in absence / presence of glucose and will be determined by MASS SPECTROMETRY.
• We detected a few proteins that are O- or N-glycosylated in Synechocystis.
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Acknowledgements
Thanks to Markéta Foldynová and MBÚ Třeboň
Thanks to Schola ludus and European social fund and budget of the Czech republic (CZ.1.07/2.3.00/09.0203)