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Šárka Cepáková, Sandra Stachová Leader: Mgr. Markéta Foldynová

Schola ludus. 28 . 07. 2011. Šárka Cepáková, Sandra Stachová Leader: Mgr. Markéta Foldynová. Protein glycosylations in cyanobacterium Synechocystis. Introduction. Subject of research - post- translational modification - glycosylation

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Šárka Cepáková, Sandra Stachová Leader: Mgr. Markéta Foldynová

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  1. Scholaludus 28. 07. 2011 Šárka Cepáková, Sandra Stachová Leader: Mgr. Markéta Foldynová Protein glycosylations in cyanobacteriumSynechocystis

  2. Introduction • Subjectofresearch - post-translationalmodification - glycosylation • Glycosylation: attachmentofglycans to proteins (N-linkedglycans, O-linkedglycans) • Model organism –  cyanobacteriumSynechocystissp. PCC 6803 • Differences in cultivation: in the presence of glucose  X autotrophiccultivation

  3. Hypothesesandaims HowdoesglucoseaffectcultivationofcyanobacteriaSynechocystis? • Effect on the biosynthesis of proteinsandpigments • Reason: alternativeglycosylation of an enzyme or regulatory factor • Expectation:  different glycosylation pattern in some proteins after cultivation in presence of glucose

  4. Methods I Isolation ofglycoproteins from cell extract: GlycoproteinIsolationKit, ConA Separation: SDS-PAGE electrophoresis Fig.1: Purificationcolumn

  5. Methods II Gel staining: • CoomassieBlueStaining • PierceGlycoproteinStainingKit Photographstaken by sensitive CCD camera

  6. Results

  7. a) Comparisonglucosecultivation X autotrophiccultivation Cell membranefraction I kDa 250 130 95 72 55 36 28 17 10 Fig.2: Autotrophiccultivation(SDS-PAGE, coomassie) Fig.3: Glucosecultivation(SDS-PAGE, coomassie)

  8. b) Comparisonglucosecultivation X autotrophiccultivation Cell membranefraction II Fig.4: Autotrophiccultivation(SDS-PAGE, coomassie) Fig.5: Glucosecultivation(SDS-PAGE, coomassie)

  9. c) Comparisonglucosecultivation X autotrophiccultivationSolubleproteins Fig.6: Autotrophiccultivation (SDS-PAGE, coomassie) Fig.7: Glucosecultivation(SDS-PAGE, coomassie)

  10. d) Comparisonglucosecultivation X autotrophiccultivation Y. H. Kim et al. (2009) Proteomics

  11. 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.

  12. Acknowledgements • Thanks to Markéta Foldynováand MBÚ Třeboň • Thanks to ScholaludusandEuropeansocialfundand budget oftheCzechrepublic (CZ.1.07/2.3.00/09.0203)

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