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Expression and purification of the Urotensin-II receptor with an affinity ligand

thesis
posted on 14.02.2017, 00:49 by Du, Ann Thi Nhu
A significant impediment to the structural and biophysical analysis of G protein-coupled receptors (GPCRs) is the inherent challenge of purifying and stabilising these receptors in an active (agonist-bound) conformation. Peptide ligands, such as the vasoactive, cyclic hormone Urotensin-II (U-II) are ideal candidates for ligand-based affinity purification of receptors due to their high affinity and tight (pseudo-irreversible) binding. The U-II receptor (UT), a receptor resistant to desensitisation, displays an unusual propensity to remain active upon ligand binding. This work aims to exploit these properties to express and purify the UT receptor at scales suitable for structural and biophysical analysis. The U-II ligand was modified with affinity groups (biotin, desthiobiotin, Strep-tag II, His6 tag) and a spacer arm at the N-terminus, creating a series of novel affinity ligands to interface with various chromatography media. The receptor-binding properties of the affinity ligands were tested (specific binding, activation and affinity). The UT receptor was stably or transiently expressed in mammalian cells (293-F or CHO-S) and the utility of the ligands were investigated during purification. Proof-of-concept studies with a biotinylated U-II peptide showed that pre-binding of affinity ligands to the UT receptor (before exposure to detergent) resulted in specific capture of UT by stabilising the receptor structure during detergent solubilisation (with n-dodecyl-β-D-maltoside). Furthermore, streptavidin-captured UT receptor/affinity ligand complexes allowed co-capture of Gαq/11 protein and were resistant to dissociation at elevated temperatures. This suggests that purified UT is able to adopt an active conformation and is relatively thermostable, making it an ideal candidate for future structural and biophysical studies. A U-II affinity ligand modified with a Strep-tag II moiety and the UT receptor modified with nine consecutive histidine residues at the C-terminus was employed for scaled expression and purification. The UT receptor was transiently expressed in 293-F cells to scales of up to 500 mL, and pre-bound ligand/receptor complexes were solubilised with n-dodecyl-β-D-maltoside and purified using immobilised metal affinity chromatography and Strep-Tactin. From 500 mL expression, 44 μg of protein was obtained with up to 75% protein recovery. This work demonstrates the value of ligand-based support of protein structure as a purification tool for GPCRs, resulting in the first successful purification of the UT receptor.

History

Principal supervisor

Marie-Isabel Aguilar

Additional supervisor 1

Walter Thomas, Leonnard Pattenden

Year of Award

2012

Department, School or Centre

Biochemistry and Molecular Biology

Campus location

Australia

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences