DOI: 10.5176/2251-2489_BioTech13.90
Authors: Ji-In Park, Jun Hyuck Lee, Yunho Gwak, EonSeon Jin, Young-Pil Kim
Abstract:
Antifreeze proteins (AFPs) have been greatly attractive due to their ability to lower the freezing temperature by inhibition of ice growth, which allows for the survival of many organisms that inhabit iceladen environments. Despite their potential applications to biotechnology and cryomedicine, a method for measuring antifreeze activity currently replies on long-lasting observations of their ice crystals at varied temperatures (thermal hysteresis, TH), which is still far from operational simplicity and rapid throughput. In the present study, we demonstrate a rapid analysis of antifreeze protein activity based on self-assembly of gold nanoparticles (AuNPs) at subzero temperature. Two different AFP-coding genes were cloned from the Antarctic diatom Chaetoceros neogracile (CnAFP) and the Arctic yeast Leucosporidium sp. (LeAFP), and their recombinant proteins were expressed in E. coli. When the AFPs were reacted to the solution of mercaptosuccnic acid-stabilized AuNPs at a cycle of 1 hfreezing and 10 min-thawing, a color change (i.e. freeze-induced aggregation) in the reddish AuNP solution was highly inhibited, as the AFP concentration increases. In contrast, there was a large aggregation and a color change into light grey in the AuNP solution
containing either control proteins (bovine serum albumin) or mutants on the ice-binding site (T65Y, S43Y, and A234Y forms in LeAFPs), probably due to the marginal inhibition of ice growth. As a result, the aggregation degree (E520/E650) of AuNPs toward the AFPs was significantly correlated with their activities (TH values). Therefore, we suggest that our developed strategy will be very suitable for measuring antifreeze activity in a simple and high-throughput way.
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