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Title

Έκφραση και απομόνωση των φυτικών λιπιδικών μεταφορασών SFH6, SFH8 και SFH9 σε ετερόλογο σύστημα E. coli και ο ρόλος τους στην κυτταρική μοίρα

Alternative Title

Expression and purification of plant lipid transferases SFH6, SFH8 and SFH9 from the heterologous system E. coli and their role in cell fate determination

Author

Χατζημιχάλη, Βασιλική

Thesis advisor

Μόσχου, Παναγιώτης
Παυλίδης Ιωάννης

Abstract

The current diploma thesis describes the methods used for the expression and purification of the plant lipid transferases, SFH6, SFH8 and SFH9 from the heterologous system Escherichia coli. Next, the purpose of the thesis was the elucidation of SFH9’s role in cell fate determination, through its interaction with common phospholipids. In order to accomplish these goals, affinity chromatography for GST and MBP tags was used, while SFH9 was later subjected to Lipid Overlay Assay. Affinity chromatography showed that, SFH6 was adequately isolated using GST purification, whereas for the isolation of the other two proteins of the SFH superfamily, MBP purification was required. This was due to the fact that, GST purification resulted in SFH9 and most of SFH8 being present only in the pellet, whereas MBP purification resulted in the proteins being present mainly in the supernatant liquid. Last but not least, Lipid Overlay Assay showed that, among 15 phospholipids, phosphatidic acid (PA) interacts strongly with SFH9 and so phosphatidylinositol 3-phosphate (PtdIns3P) does, but in a lower degree.

Language

Greek

Subject

GST purification

Lipid Overlay Assay

MBP purification

Phosphatidic acid

Plant lipid transferases

SFH superfamily

Απομόνωση με GST

Απομόνωση με MBP

Οικογένεια SFH