Engineering Antifreeze Protein Multimers Through Genetic Fusion to Self-Assembling Protein Oligomers
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Ice-binding proteins (IBPs) serve many purposes for the organisms producing them, enabling them to thrive in ice-laden environments. Antifreeze proteins (AFPs), a subset of this group, act as biological antifreezes and inhibit ice recrystallization. These ice-binding activities have many applications in the field of biotechnology, and to this end AFPs have been engineered in several ways to increase their activity. One of these strategies is to connect multiple AFPs together using a scaffold, which has been accomplished previously through bioconjugation of AFPs to the reactive termini of branched polymers. The inefficiency of such linkage reactions, however, resulted in heterogeneous mixtures of particles with incomplete occupancy of the available termini. To address this shortcoming, AFPs were genetically fused to the C termini of subunits forming self-assembling protein oligomers, in such a way that the ice-binding site of the AFP was optimally exposed for contact with ice. First, two different type of AFPs were separately fused to 12 of the 24 subunits of a self-assembling protein cage. The subunits assembled into the designed structures, and the AFP multimers showed greatly enhanced freezing point depression and ice recrystallization inhibition over the corresponding monomeric AFPs. A moderately active fish AFP was also fused to a subunit designed to assemble into two-dimensional arrays, and the resulting fusion protein showed increased freezing point depression over the monomer. However, size-exclusion chromatography of the fusion protein suggested that the subunits were not assembled into a two-dimensional array, and additional structural characterization is needed to elucidate the oligomerization state of this design. Overall, genetic fusion of AFPs to the termini of self-assembling oligomers has proven to be a promising method of enhancing their ice-binding activities, and could be used to arrange AFPs into novel orientations. We also envision that the RosettaDesign program used to design the novel protein assemblies can be applied to AFPs themselves, to generate AFP-to-AFP binding sites on their surfaces and arrange them into oligomers not seen in nature.
URI for this recordhttp://hdl.handle.net/1974/22612
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