The traditional anti-icing/deicing agents are propylene or ethylene glycol. Glycols are effective in lowering the freezing point of water mixtures by the phenomenon of freezing point depression which is based solely on the molal concentration. The key environmental concerns with respect to the use of ice control fluids are biological oxygen demand (BOD) loading and toxicity (human/mammalian and aquatic) which result in extensive collection and cleanup costs.
This project proposes to genetically alter the Dendroides canadensis antifreeze protein gene in order to enhance its freezing point depression capabilities and increase its usefulness and value as an aircraft anti-icing agent.
Initially, site-directed mutagenesis of the D. canadensis antifreeze protein (AFP) gene will be completed. Select amino acids within the ice crystal binding domain will be changed to enhance the antifreeze activity of the D. canadensis protein, and DNA sequence analysis will be performed of the mutant D. canadensis genes.
After the cloning and selection of specific site-directed mutagenesis mutants of the gene have been completed, the DNAs will be sequenced. Cloning, selection, and analysis of the mutant genes will be conducted. Then, the constructed gene for the mutated D. canadensis AFP will be ligated into the pHIL-S1 yeast expression vector containing the Pichia signal sequence, and expression of the mutant D. canadensis AFP will occur. A single colony from each clone of interest will be inoculated, incubated, and centrifuged before the supernatant is collected for analysis. The mutant D. canadensis AFP then will be purified.
The expanded bed absorption chromatography technology (STREAMLINE) developed by Pharmacia Inc. will be used to purify the mutant AFP from the crude fermentation media and cells. The antifreeze capabilities will be determinations of mutant D. canadensis AFPs. Recrystallization inhibition properties of potential antifreezeagents will be determined using a splat-cooling method. In addition, BOD, toxicity, and environmental fate testing of the mutant D. canadensis statement of the ecological behavior for each of the mutant AFPs will be conducted. A D. canadensis AFPs that are produced during the course of this program then will be produced. Finally, the scale-up production of the mutant D. canadensis AFPs will be initiated.
The accomplishments to date include the amino acid sequence analysis and binding domain comparisons of the Dendriodes canadensis antifreeze protein with other published AFP sequences. The design, synthesis, and conformational sequencing of the mutagenesis DNA oligonucleotides to be used to mutate the D. canadensis AFP gene have been conducted. The cloning of the gene’s DNA into the pAlter II mutagenesis cloning vector has been completed, as has the confirmation of the mutated sequences by DNA sequencing. The cloning of the mutant D. canadensis AFP genes into the yeast, Pichia pastoris, and their confirmation by PCR analysis was made. Three of these clones have since been proven to express an immunoreactive protein that is secreted into the media which confirms the presence of an AFP.
A successful program will push the development of antifreeze proteins as anti-icing agents to enable their application in the field, replacing environmentally unacceptable materials such as propylene glycol and urea in aircraft and runway ground anti-icing fluids and solids.