Abstract
Adaptation to cooler climates is a necessity for the organisms which endure temperatures below freezing. The yellow mealworm beetle, Tenebrio molitor, overwinters in the larval stage and is therefore succeptable to freezing conditions. To reduce the risk of freezing, which can be lethal, T. molitor express thermal hysteresis protein (THP) into their haemolymph that bind to the forming surface of ice crystals and lower the freezing temperature of the organism’s fluids, which allows the larvae to survive temperatures down to −13 ○C. This small, highly disulfide bonded protein is traditionally produced recombinantly for analysis. However there are cleavages in the peptide backbone in the native protein that E. coli cannot produce and potentially play a role in the function. This research outlines the purification of THP from both the T. molitor larvae and a recombinant expression system and the two samples were tested using optical recrystallometry and nanolitre osmometry. The data show the two sources to have similar recrystallometry inhibition activity, however the native protein proves to be more active in reducing the freezing temperature, and therefore increasing the thermal hysteresis, than the recombinant protein. The data also show evidence of the thermal hysteresis proteins forming dimers and other multimers and suggests there are interactions between the many isoforms produced by T. molitor. The difference in activity between the two sources shows the importance of understanding post-translational modifications when using a recombinant expression system and knowing how proteins function in relation to other proteins in vivo.