The Library
Expression and purification of the plant AUX1 auxin-influx carrier protein for structural characterization
Tools
Joshi, Chitra (2022) Expression and purification of the plant AUX1 auxin-influx carrier protein for structural characterization. PhD thesis, University of Warwick.
PDF
WRAP_THESIS_Joshi_2022.pdf - Submitted Version Embargoed item. Restricted access to Repository staff only until 3 March 2025. Contact author directly, specifying your specific needs. - Requires a PDF viewer. Download (63Mb) |
Official URL: http://webcat.warwick.ac.uk/record=b390215
Abstract
The phytohormone auxin regulates myriads of developmental processes and is unique for exhibiting polar transport. Indole-3-acetic acid (IAA) a major form of auxin is a weak acid, and the polarity of auxin movement is facilitated by asymmetric localization of influx and efflux membrane proteins. AUXIN1/LIKE-AUX1 (AUX1/LAX) proteins form a plant-specific sub-class within the amino acid/auxin permease super family, encoding auxin uptake carriers. Despite being a master player in auxin homeostasis, the molecular mechanism of AUX1/LAX proteins remains poorly understood. Therefore, the aim of my PhD project was to determine the mechanism of auxin accumulation by delineating an atomic structure of AUX1 through X-ray crystallography. To provide enough protein for biochemical studies, I engineered the expression of AUX1/LAX homologues and developed an optimized system for the heterologous expression of AUX1 in Sf9 insect cell system using a highthroughput platform in collaboration with the Membrane Protein Laboratory at Diamond Light Source, UK. The truncated version of Brachypodium distachyon AUX1 (S54-G466) was chosen for protein purification and crystallography. Purification using affinity and size exclusion chromatography was optimized at Warwick to obtain sufficient yields of the purified protein for crystallography. Several trials using vapor diffusion and lipidic cubic phase methods were carried out for obtaining protein crystals. However, AUX1 has been found to be a difficult protein to crystallize owing, in part, to instability at higher protein concentrations. Therefore, a pathway for structural determination using cryo- EM was laid by screening conditions for sample preparation for cryo-grids. A low resolution 2D classification has been obtained in collaboration with MPL. In parallel, in-silico homology modelling and functional assays using whole-cell based electrophysiology, SPR and SSM-based electrophysiology were carried out to provide hints on structure-function features of AUX1. Furthermore, funding from INSTRUCT-ERIC was secured for nanobody discovery for which protein samples have been sent to VIB, Brussels. The outcome of the project has been the development of an optimized system for heterologous expression and purification of AUX1 protein which has created a foundation for the future work towards structure-function characterization.
Item Type: | Thesis (PhD) | ||||
---|---|---|---|---|---|
Subjects: | Q Science > QD Chemistry Q Science > QK Botany Q Science > QP Physiology |
||||
Library of Congress Subject Headings (LCSH): | Auxin, Plant hormones, Membrane proteins, Carrier proteins, Crystallography | ||||
Official Date: | September 2022 | ||||
Dates: |
|
||||
Institution: | University of Warwick | ||||
Theses Department: | School of Life Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Napier, R. (Richard) ; Cameron, Alexander | ||||
Sponsors: | Commonwealth Scholarship Commission in the United Kingdom ; Medical and Life Science Research Fund ; Instruct-ERIC | ||||
Format of File: | |||||
Extent: | xix, 252 pages : illustrations (colour) | ||||
Language: | eng |
Request changes or add full text files to a record
Repository staff actions (login required)
View Item |