Graduation Year

2021

Document Type

Thesis

Degree

M.S.

Degree Name

Master of Science (M.S.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Libin Ye, Ph.D.

Committee Member

Gary Daughdrill, Ph.D.

Committee Member

Jianfeng Cai, Ph.D.

Keywords

Pichia pastoris, 19F NMR, Purification, A2AR

Abstract

GPCR regulates numerous diverse physiological processes relevant to diabetes, obesity, Alzheimer's diseases, and several central nervous system disorders and targets proteins in signaling pathways. It has created nearly 200 billion profits from its derivative drugs in 2018. There are near 400 structures of over 70 GPCRs have been resolved by X-ray crystallography, cryo-electron microscopy, and NMR spectroscopy. One of the current challenges that remain in the conformational transition and dynamics study using NMR spectroscopy is to obtain sufficient quantities of the G proteins and GPCRs. Pichia pastoris has shown its tremendous promise in expressing the GPCRs in a high yield, along with isotopic labeling capacity for NMR studies. However, additional work is needed to reach the quantity requirement for most of the receptors. On the other hand, the mammalian system and insect cell expression systems require a higher cost of producing G proteins and GPCRs and it is still challenging for isotopic labeling. In this regard, this thesis is focused on developing and optimizing an applicable P. pastoris expression system for G proteins particularly to reach certain quantities required for NMR studies. In the meantime, a fluorine-19 screening was also performed to search for a next-generation fluorine-19 tag in probing GPCR conformational states at a higher resolution than the current state-of-the-art probe BTFMA. Through two-year work, we successfully established platforms for preparing yeast-derived G proteins, including G[U+F061] and G, which should be sufficient to conduct NMR studies. Though we are still exploring a next-generation 19F probe, the current screening has given us several promising hits.

Download

Share

COinS