Iodine plays a key role in many organisms. It is essential micro-nutrient for humans and animals, but its role in plants remains relatively unknown. Iodine deficiency in humans can cause numerous health issues, hence scientists are striving to find ways to limit its impact. Biofortification of crops is very promising, but understanding the role of iodine in plants is crucial for its effective implementation. One of the unresearched areas are iodinated proteins identified in the leaves and roots of A. thaliana. These include Ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RuBisCO activase) and Fructose-bisphosphate aldolase 2 (AtFBA2), both playing important roles in the Calvin cycle and are essential for various photosynthetic organisms. With the aim of discovering the role of iodine in RuBisCO activase and AtFBA2, we begun the process of molecular cloning and preparation of recombinant proteins. We successfully prepared the sequences for both selected proteins. After the addition of N-terminal hexahistidine tag and a recognition site for TEV protease, we transferred them into the pET-32b(+) vector. We successfully transformed E. coli DH5α cells, followed by plasmid isolation and its insertion into expression strain. We continued with expression and isolation using Ni-affinity chromatography followed by the gel application and analysis of the obtained results. Because of our unsuccessful expression, we could not proceed with studying the function of iodine in the selected proteins. Thus, the process we used will need further optimization.
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