Recombinant expression of natural Arabidopsis PRLIP1 variants reveals temperature-dependent differences in fluorescence and functional protein recovery in Escherichia coli.

Recombinant protein production in Escherichia coli is frequently limited by aggregation into inclusion bodies, reducing the recovery of active protein. In this study, we compared two naturally occurring Arabidopsis thaliana PRLIP1 variants from the Columbia-0 and Wassilewskija accessions, which differ by 13 amino acids, during temperature-controlled heterologous expression as GFP fusion proteins. GFP fluorescence, protein distribution between soluble and insoluble fractions, and esterase activity recovered after alkaline solubilization and refolding were analysed. Reduced cultivation temperatures increased GFP fluorescence and recovered esterase activity, although most of the recombinant protein still remained associated with inclusion bodies. Differences between the two PRLIP1 variants were observed in fluorescence intensity, inclusion body accumulation, and recovered esterase activity, indicating that natural PRLIP1 variation is associated with altered recombinant protein behaviour. Sequence-based hydropathy analysis revealed no major global differences in overall hydrophobicity, whereas aggregation prediction indicated subtle local differences in aggregation-prone regions. These results suggest that naturally occurring allelic variation can provide a useful first-pass approach to identify sequence-associated differences in folding-related behaviour and functional recovery, while residue-level causal mechanisms require further mutational analysis.