Optimisation of Ramonda serbica LEA protein production in Escherichia coli and its secondary structure analysis

Desiccation, an extreme form of dehydration, reduces the cellular water content to below 5 % and poses a major challenge for most plants. Ramonda serbica, a tertiary relict and homoiochlorophyllous resurrection plant, is an exceptional model for investigating vegetative desiccation tolerance. Late Embryogenesis Abundant (LEA) proteins are strongly involved in this adaptive trait, but their exact molecular function is still unclear. In this study, we report the first successful recombinant production of the desiccation-induced LEA protein, RsLEAP30, from a dicotyledonous resurrection plant species using an Escherichia coli expression system. By employing immobilised metal affinity and size-exclusion chromatography, we achieved to purify RsLEAP30 to purity over 95 %, providing a robust and scalable method for producing other LEA proteins. Structural characterisation by circular dichroism spectroscopy, combined with in silico modelling, revealed that RsLEAP30 is predominantly disordered but contains -helical regions. We suggest that this structural duality underpins the protective role of RsLEAP30 in chloroplasts, likely via interactions with thylakoids and desiccation-sensitive proteins within photosynthesis-associated proteins. This function may be crucial for the rapid recovery of photosynthetic components upon rehydration. Our study provides new insights into the structure-function relationship of LEA proteins in resurrection plants and establishes a foundation for future investigations. Understanding the protective mechanisms of RsLEAP30 will pave the way for bioengineering strategies aimed at improving the drought tolerance of crops.