Activity and inhibitory study of reconstituted Lyp1 from Saccharomyces cerevisiae

Fang, Biqin (2021) Activity and inhibitory study of reconstituted Lyp1 from Saccharomyces cerevisiae. Bachelor's Thesis, Chemistry.

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Abstract

Lyp1 is a lysine permease from the SLC12A transporter family, which is also a unidirectional secondary transporter from Saccharomyces cerevisiae. It has 12 trans-membrane spanning segments with both N- and C-terminus inside the plasma membrane. Lyp1 equipped with a GFP tag was heterologously expressed in Pichia pastoris with methanol as the induction agent. 14C-lysine uptake assay is the main reconstituted Lyp1 activity and inhibitory measurement. Since the exact topological three-dimensional structure of Lyp1 has not been revealed yet, we purified and reconstituted Lyp1 into liposomes containing different ergosterol mole percentages; among three ergosterol percentages, 10 mol percent achieved the highest activity. Because of the different affinity of the two sides of Lyp1, this lysine-specific permease preferably transports lysine from outside of the cell to inside. As possible inhibitors, lysine and arginine analogues, antidepressants and natamycin were tested at 20 to million-fold excess to lysine. By measuring the counts of radio activit, we observed full inhibition with a high concentration of 100 mM L-ornithine but also lower concentration of 20 μM and 200 μM thialysine. We then found the relative potential structural stabilizer of Lyp1.

Item Type:	Thesis (Bachelor's Thesis)
Supervisor name:	Obermaier, S. and Poolman, B. and Slotboom, D.J.
Degree programme:	Chemistry
Thesis type:	Bachelor's Thesis
Language:	English
Date Deposited:	12 Jul 2021 10:04
Last Modified:	12 Jul 2021 10:04
URI:	https://fse.studenttheses.ub.rug.nl/id/eprint/25165

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