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Expression and purification of the auxin efflux transporter PIN1 from the arabidopsis PIN protein family in Sf9 insect cells
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Zhang, Fangfang (2019) Expression and purification of the auxin efflux transporter PIN1 from the arabidopsis PIN protein family in Sf9 insect cells. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3493245~S15
Abstract
The auxin efflux carrier PIN-FORMED 1 (PIN1) protein plays a crucial role in polar auxin transport in plants as its asymmetrical localization in cells determines the direction of cell-to-cell auxin flow. However, despite decades of investigation, the successful high-yield purification of PIN1 has not been reported. To understand the structure and function mechanism of PIN1 in polar auxin transport, we developed a high-level heterologous expression system using recombinant baculoviruses and Spodoptera frugiperda (Sf9) insect cells for the production of Arabidopsis full-length PIN1, PIN1-CUT, and PIN5 (loop-truncated PIN1). We chacterized PIN1, PIN1_CUT, and PIN5 by immunoblotting as well as in-gel fluorescence detetion. Confocal microsopy confirmed the membrane-associated subcelullar localization of the three PIN proteins, indicating intact protein production by Sf9 cells. Detergent screening was conducted by the fluorescence-detection size-exclusion chromatography (FSEC) analysis. Based on the FSEC results, we selected n-Dodecyl-N,NDimethylamine- N-Oxide (LDAO) and n-Dodecyl β-D-maltoside (DDM) as optimized protein extraction and immunoaffinity purification detergents. Buffer pH values between 7.4 and 8.0 without the presence of reducing agents were also observed to have a positive effect on deminishing protein aggregates. PIN1_CUT purification was optimized using anti-FLAG M2 affinity resin and approciately 40% PIN1_CUT proteins were recovered from DDM-solubilized protein samples. The overall PIN1_CUT purification yield from whole cell lysate samples was close to 15%. We thus succeeded in overcoming the heterologous expression and purification of PIN1_CUT, laying a solid fundation for the future crystallographic studies.
| Item Type: | Thesis (PhD) | ||||
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| Subjects: | Q Science > QK Botany Q Science > QL Zoology |
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| Library of Congress Subject Headings (LCSH): | Auxin, Arabidopsis, Fall armyworm, Plant hormones | ||||
| Official Date: | June 2019 | ||||
| Dates: |
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| Institution: | University of Warwick | ||||
| Theses Department: | School of Life Sciences | ||||
| Thesis Type: | PhD | ||||
| Publication Status: | Unpublished | ||||
| Supervisor(s)/Advisor: | Napier, R. (Richard) ; Cameron, Alexander | ||||
| Format of File: | |||||
| Extent: | xvi, 167 leaves : illustrations | ||||
| Language: | eng |
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