Insect antifreeze proteins and the expression and characterization of spruce budworm isoforms in transgenic systems

Antifreeze proteins (AFP) are important for freezing point depression in 2nd instar Choristonettra fumiferana, spruce budworm larvae. A total of 14 sequences encoding several AFP isoforms were cloned and characterized. The importance of specific residues in the structure and function of C. fumiferana AFP was assessed by examining closely related species. The sequences encoding the conserved beta-helix region were subcloned from approximately one-third of the AFP genes from 3 sister budworm species. The aligned sequences show a high conservation of functional residues, including threonines in the ice-binding face and residues important for structure such as cysteine and seine. Divergence analysis dated the AFP gene duplication event to ∼4 million years ago, close to the beginning of recent glaciation events in North America.; In spruce budworm transcripts were highest in diapausing 2nd instars, but messages encoding some isoforms were also detected in eggs, 1 st instars, and in 6th instar gut tissue. Transcript levels did not appear to be affected by the tested temperatures or by altering the photoperiod, suggesting that AFP expression is developmentally regulated, and not influenced by environmental factors. Investigation of AFP transcript abundance in 6th instars showed low levels in gut tissue with AFP detected in gut contents and not in the hemolymph, as in diapausing instars. One novel isoform, Cf6, shows a high degree of sequence identity to a previously characterized isoform, Cf10, but has 2 adjacent disulfide bonds disrupted by mutations. Refolding of Cf6 and Cf10 from bacterial inclusion bodies show recoverable activity for only Cf10.; Heterologous expression systems were explored as a means to produce large quantities of individual AFP isoforms. Expression in Pichia yeast resulted in extensive glycosylation of the AFP, reducing the activity 99% when expressed in large-scale fermentors. Heterologous expression in transgenic Drosophila gave better results with properly folded, active AFP secreted into the hemolymph. While variable AFP activity was observed between fly lines, the highest recorded value of 0.8°C thermal hysteresis did not confer cold tolerance to the flies at low non-freezing temperatures. Heterologous expression of several isoforms in Ori-DE3 E. coli bacteria resulted in low-level expression of soluble proteins that lacked AFP activity and were likely misfolded. Induction of several budworm AFPs in another E. coli host strain, BL21-DE3, resulted in protein sequestration into inclusion bodies, resolublizing and refolding inclusion body protein was largely unsuccessful with only a fraction of the refolded protein staying soluble and having activity. This research has not only increased our understanding of these novel proteins, their evolution, their expression and their structure, but has also underscored the challenges of the study of highly folded, hyperactive antifreeze proteins.