Functional evaluation of candidate ice structuring proteins using cell-free expression systems

► We describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status. ► 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 desc...

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Bibliographic Details
Published in:	Journal of biotechnology Vol. 163; no. 3; pp. 301 - 310
Main Authors:	Brödel, A.K., Raymond, J.A., Duman, J.G., Bier, F.F., Kubick, S.
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 10-02-2013
Subjects:	Animals Antifreeze protein Antifreeze Proteins - metabolism Cell-free protein synthesis Cell-Free System - metabolism Chlamydomonas - metabolism Crystallization Fishes - metabolism Freezing Glycosylation Ice Ice binding protein Ice structuring protein In vitro translation Insecta - metabolism Water - metabolism In vitro translation Ice binding protein Antifreeze protein Cell-free protein synthesis Ice structuring protein
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Summary:	► We describe cell-free expression methods for three ISPs that differ widely in structure and glycosylation status. ► 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. 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0168-1656 1873-4863
DOI:	10.1016/j.jbiotec.2012.11.001