Evaluation of different strategies to produce recombinant Arabidopsis thaliana SOG1

Student thesis: Master's Thesis

Abstract

Plants are everywhere. But due to their sessile lifestyle, they are very susceptible to all types of environmental stresses. These can cause many types of DNA damage to the genome and interfere with the normal integrity of the cells and the organism. Luckily, special pathways exist to monitor and remedy the DNA damage. The most studied one in eukaryotes is the so-called DNA Damage Response Pathway. The signal cascade all comes together into a central, master regulator. This master regulator in plants is called SOG1, or “Suppressor of Gamma Response 1”. SOG1 consists of a highly structured DNA binding domain, the NAC domain, and a intrinsically disordered regulatory domain. Earlier attempts to purify SOG1 from posed many problems in terms of purity and functionality of the protein.
This thesis is focused on the comparison and optimisation of different purification strategies to purify SOG1 via a prokaryotic E. coli expression system. A purification strategy to purify from inclusion bodies instead of the soluble phase was developed: Unfolding-Refolding on Column purification strategy. This strategy was first tested on NAC3, a construct of SOG1 that only contains the highly structured NAC domain. This construct can also be purified from the soluble phase. By comparing both the “Refolded NAC3” and the “Native NAC3”, it was verified if the folded part of SOG1 could refold back to its native state. If this was possible, the strategy would be viable for a full length SOG1 purification. Both NAC3 purification strategies were optimised. The overall yield of the purification from the soluble phase was increased to around 66 mg per litre of culture, and the Unfolding Refolding on column strategy to 0,67 mg per litre of culture. The resulting protein from both strategies was compared using different functional characterisation techniques. DLS showed no notable difference in hydrodynamic radius. No difference in DNA binding capabilities was noted using EMSA. The yield of the Unfolding-Refolding Protocol was too low to perform ITC and Analytic SEC. With the remaining NAC3 sample, an attempt to crystallize the protein was made, but no crystals formed as of writing this thesis. The different techniques showed no notable difference between the Refolded NAC3 and the Native NAC3, so the Unfolding-Refolding on Column protocol was attempted for the purification of the full length SOG1 construct. After trying the protocol several times, no SOG1 could be purified using this protocol as it was developed during this thesis. K. Mignon developed a strategy to purify SOG1 from the soluble phase using a SOG1 construct with a C-terminal SUMO solubility tag. The yield using this technique was 3,2 mg per litre of culture. Functional characterisation techniques proved the SOG1 protein was functional. The EMSA showed that the purified SOG1 did bind with a known target DNA sequence. Analytic SEC and DLS showed a molecular mass and hydrodynamic radius as expected for SOG1 with its IDR.
This thesis has laid the groundwork for the characterisation and further studies of SOG1. The further study of SOG1 will provide insights to how plants react to DNA damage. Considering the ever changing climate and pollution, these insights may become crucial to improve the viability of plants in the future.
Date of Award11 Sep 2023
Original languageEnglish
Awarding Institution
  • Department of Bio-engineering Sciences
  • Vrije Universiteit Brussel
SupervisorMargot Galle (Advisor) & Remy Loris (Promotor)

Keywords

  • DNA damage response
  • DNA binding
  • transcription factor
  • protein purification
  • molecular biophysics

Cite this

'