Antifreeze Protein Engineering

• Main Author: Stevens, Corey
• Other Authors: Davies, Peter, Biochemistry
• Format: Thesis
• Language: English
• Published: 2017
• Subjects: Antifreeze Protein; Protein Engineering; Adhesion Protein; Adhesin; Diatom; Microfluidics; Ice-Binding Protein; Dendrimer; Intein; Protein Circularization; Thermal Stability; Protein Multimers; Marinomonas Primoryensis; Chaetoceros Neogracile; Peptide Backbone Circularization; Antarctic; The Antarctic; Antarc*
• Online Access: http://hdl.handle.net/1974/22711

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. PhD