• search hit 1 of 1
Back to Result List

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

  • 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 prokaryoticIce 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.‚Ķshow moreshow less

Export metadata

Additional Services

Share in Twitter Search Google Scholar Statistics
Metadaten
Author:A. K. Broedel, J. A. Raymond, J. G. Duman, Frank Fabian BierORCiDGND, S. Kubick
DOI:https://doi.org/10.1016/j.jbiotec.2012.11.001
ISSN:0168-1656 (print)
Parent Title (English):JOURNAL OF BIOTECHNOLOGY
Publisher:ELSEVIER SCIENCE BV
Place of publication:AMSTERDAM
Document Type:Article
Language:English
Year of first Publication:2013
Year of Completion:2013
Release Date:2017/03/26
Tag:Antifreeze protein; Cell-free protein synthesis; Ice binding protein; Ice structuring protein; In vitro translation
Volume:163
Issue:3
Pagenumber:10
First Page:301
Last Page:310
Funder:German Ministry of Education and Research (BMBF) [0312039]