Molecular Cloning and Overexpression of Arabidopsis thaliana 14-3-3𝛋 Protein in Escherichia coli

Abstract

The 14-3-3 protein family plays a crucial role in regulating multiple physiological pathways in plants, particularly those involved in responses to both biotic and abiotic stresses. In Arabidopsis, 14-3-3 proteins interact with key partners to modulate signalling pathways, particularly those involved in stress tolerance. Besides, Ca²⁺ signalling in plants regulates plant transporters' activities in stress tolerance. However, Ca²⁺ influence on 14-3-3 function, stability, and binding affinity remains unclear. To explore this, we performed bioinformatic analyses of one of the Arabidopsis thaliana 14-3-3 variants, 14-3-3𝛋, which is known to bind plant plasma membrane SOS1 C-terminal residues. Since SOS1 is also indirectly regulated by cytosolic Ca²⁺, we aimed to investigate whether Ca²⁺ influences the structure, function, or binding affinity of 14-3-3𝛋. Arabidopsis thaliana 14-3-3 sequences and structures are studied and compared with the known peptide-bound 14-3-3 structures to explore this. The cloning was accompanied by the overexpression of 14-3-3𝛋 in E. coli following affinity purification. Future biochemical characterisation will focus on understanding the interaction between calcium and 14-3-3. This study may provide insights into the broader role of 14-3-3 and its interaction with Ca²⁺, which can be used in developing stress-resilient crops.