Expression and purification of arabidopsis thaliana heterotrimeric G protein alpha subunit (GPA1) using bacteria and yeast systems

Köse, Hazal Büşra (2014) Expression and purification of arabidopsis thaliana heterotrimeric G protein alpha subunit (GPA1) using bacteria and yeast systems. [Thesis]

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Heterotrimeric G-proteins mediate transmission of signals from G-protein coupled receptors to cell interior and activate signaling pathways in several organisms from yeast to mammals and plants. The heterotrimer consists of the alpha (Gα), beta (Gβ) and gamma (Gγ) subunits; Gα has GTP binding and hydrolysis activity and Gα and Gβ/Gγ dimeric complex interact with downstream effectors upon activation. G-protein subunits have been identified in several plant species and were shown to be involved in growth and in responses to light and environmental stress factors including draught and pathogens [1]. Although the crystal structure data at 2.34 Å resolution is available for A. Thaliana Gα [2] but direct structural data on the Gβ/Gγ dimer and the heterotrimer as a whole are lacking in the literature. The mechanism of activation for the plant heterotrimer is generally inferred by assuming analogy with the mammalian complex. However, recent studies indicate that the plant α subunit may possess a self activation mechanism not observed in the mammalian system which sets a limit to the extent of the analogy [3]. Previously in our group we cloned and expressed Arabidopsis thaliana α subunit (GPA1) using Pichia Pastoris and β (AGB1) andγ (AGG2) subunits using E.coli systems [4, 5] with the aim structure-function studies on the individual subunits and the reconstituted complex. However, since expression and purification of the recombinant protein from yeast are more time and resource consuming compared with E.coli, we investigated possibilities for cloning the GPA1 sequence using alternative methods. In this study, results of cloning of GPA1 using pETM41 and pQE80L vectors are given together with investigation of expression. In different E.coli host cells, after optimizing the GPA1 expression, recombinant GPA1 was purified and biochemically characterized. Preliminary structural characterization of E.coli produced GPA1 was also conducted by circular dichroism spectropolarimetry and dynamic light scattering measurements. Results show that GPA1 can be expressed in the Rosetta strain of E.coli as a fusion with maltose binding protein (MBP) giving a yield of 1.2 mg of protein from 1 L of culture. However, during cleavage of MBP tag with tobacco etch virus (TEV) protease, precipitation results in the loss of more than 70% of the purified protein with the remaining part of GPA1 being aggregated. We suggest that the one possible cause of precipitation is the N-terminal flexible region of GPA1 consisting of 36 aminoacids which may disrupt the stability. Another possible reason is the lack of post-translational modifications in E.coli. We did not observe any aggregation in GPA1 samples produced in P.pastoris system. Structural characterization experiments showed that the secondary structure content of P.pastoris GPA1 is consistent with the crystal structure data of mammalian GPA1. Further experiments are required to both improve the purification results to produce more GPA1 and for structural characterization of the protein.
Item Type: Thesis
Uncontrolled Keywords: G proteins. -- GPA1. -- Different expression systems. -- G proteinleri. -- GPA1. -- Farklı ekspresyon sistemleri.
Subjects: T Technology > TA Engineering (General). Civil engineering (General) > TA164 Bioengineering
Divisions: Faculty of Engineering and Natural Sciences > Academic programs > Biological Sciences & Bio Eng.
Faculty of Engineering and Natural Sciences
Depositing User: IC-Cataloging
Date Deposited: 25 Apr 2018 16:39
Last Modified: 26 Apr 2022 10:18

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