TY - JOUR A1 - Broedel, A. K. A1 - Raymond, J. A. A1 - Duman, J. G. A1 - Bier, Frank Fabian A1 - Kubick, S. T1 - Functional evaluation of candidate ice structuring proteins using cell-free expression systems JF - JOURNAL OF BIOTECHNOLOGY N2 - Ice structuring proteins (ISPs) protect organisms from damage or death by freezing. They depress the non-equilibrium freezing point of water and prevent recrystallization, probably by binding to the surface of ice crystals. Many ISPs have been described and it is likely that many more exist in nature that have not yet been identified. ISPs come in many forms and thus cannot be reliably identified by their structure or consensus ice-binding motifs. Recombinant protein expression is the gold standard for proving the activity of a candidate ISP. Among existing expression systems, cell-free protein expression is the simplest and gives the fastest access to the protein of interest, but selection of the appropriate cell-free expression system is crucial for functionality. Here we describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status from three organisms: a fish (Macrozoarces americanus), an insect (Dendroides canadensis) and an alga (Chlamydomonas sp. CCMP681). We use both prokaryotic and eukaryotic expression systems for the production of ISPs. An ice recrystallization inhibition assay is used to test functionality. The techniques described here should improve the success of cell-free expression of ISPs in future applications. (C) 2012 Elsevier B.V. All rights reserved. KW - Ice structuring protein KW - Antifreeze protein KW - Ice binding protein KW - Cell-free protein synthesis KW - In vitro translation Y1 - 2013 U6 - https://doi.org/10.1016/j.jbiotec.2012.11.001 SN - 0168-1656 VL - 163 IS - 3 SP - 301 EP - 310 PB - ELSEVIER SCIENCE BV CY - AMSTERDAM ER -