Antifreeze Protein Engineering
This thesis describes: 1) the use of protein engineering to increase ice-binding protein (IBP) activity and thermal stability, and 2) the binding interaction and microcolony formation between an Antarctic bacterium and diatom. IBPs, including the antifreeze proteins (AFPs) that prevent the freezing of organisms, are found in nearly all biological kingdoms. IBPs have potential applications in a variety of domains including the food industry, cryo-medicine, and biotechnology. Despite the variety of IBPs, most are difficult to produce in amounts needed for industrial applications. Consequently, there is a need to find or engineer IBPs with enhanced activity and stability. Previously, AFP activity was increased by fusing an AFP to another protein, or by increasing the size of the IBP’s ice-binding face. Here, I used a highly-branched polymer, known as a dendrimer, to fuse a range (6 to 12) of moderately-active type III AFPs from Macrozoarces americanus together. These AFP multimers had improved antifreeze and ice-recrystallization inhibition activity. Unexpectedly, AFPs multimers had enhanced recovery from heat treatment. I also achieved enhanced thermal stability in type III AFP through an alternative strategy. Using split-intein mediated end-terminal ligation, I fused the N- and C- termini of the type III AFP together. Peptide backbone circularization had no effect on antifreeze activity but significantly increased thermal stability compared to the non-cyclized form. The IBP found on the cell surface of a Gram-negative Antarctic bacterium, Marinomonas primoryensis, is one region of an exceptionally large multi-domain 1.5 MDa protein, MpIBP. Using temperature-controlled microfluidics, I have shown that M. primoryensis forms bacterial clusters on ice. Binding is aided by the motility of the bacterium and is dependent on the functionality of its ice-binding domain. The strictly aerobic M. primoryensis is drawn and binds to, the Antarctic diatom Chaetoceros neogracile to form mixed cell clusters and adheres them to ice. We hypothesize that the ice-binding function of MpIBP keeps its host immediately under the surface ice in the phototrophic zone of the water column where oxygen and carbon compounds are more abundant. By recruiting diatoms to the ice, M. primoryensis helps these photosynthesizers form a symbiotic community where light is most abundant.
Request an alternative formatIf you require this document in an alternate, accessible format, please contact the Queen's Adaptive Technology Centre
The following license files are associated with this item:
Except where otherwise noted, this item's license is described as Queen's University's Thesis/Dissertation Non-Exclusive License for Deposit to QSpace and Library and Archives Canada
Showing items related by title, author, creator and subject.
Biochemical Characterization of Nucleotide and Protein Interactions of Human Multidrug Resistance Protein 1 (MRP1/ABCC1) Wang, Xiaoqian (2008-12-09)Multidrug resistance protein 1 (MRP1) is an integral membrane protein belonging to the ATP-binding cassette (ABC) superfamily that utilizes ATP binding and hydrolysis to transport various endogenous substrates and/or ...
PROTEIN KINASE A, EXCHANGE PROTEIN ACTIVATED BY cAMP 1, AND PHOSPHODIESTERASE 4D ALL ASSOCIATE WITH VE-CADHERIN TO REGULATE ENDOTHELIAL BARRIER FUNCTION Ovens, Jeffrey David (2007-09-17)Vascular endothelial cells (VECs) play an essential role in regulating the passage of macromolecules and cells between the blood stream and underlying tissues. The second messenger 3’, 5’ cyclic adenosine monophosphate ...
Regulation of the Human Ether-a-go-go Related Gene Potassium Channel by Neural Precursor Cell Expressed Developmentally Down-regulated Protein 4-2 Interacting Proteins Kang, Yudi (2015-08-19)Dysfunction of the human ether-a-go-go related gene (hERG)-encoded rapidly activating delayed rectifier K+ channel is a major cause of long QT syndrome (LQTS) due to its critical role in the repolarization of cardiac action ...