TY  - JOUR
A1  - Sachse, Rita
A1  - Wüstenhagen, Doreen Anja
A1  - Samalikova, Maria
A1  - Gerrits, Michael
A1  - Bier, Frank Fabian
A1  - Kubick, Stefan
T1  - Synthesis of membrane proteins in eukaryotic cell-free systems
JF  - Engineering in life sciences : Industry, Environment, Plant, Food
N2  - Cell-free protein synthesis (CFPS) is a valuable method for the fast expression of difficult-to-express proteins as well as posttranslationally modified proteins. Since cell-free systems circumvent possible cytotoxic effects caused by protein overexpression in living cells, they significantly enlarge the scale and variety of proteins that can be characterized. We demonstrate the high potential of eukaryotic CFPS to express various types of membrane proteins covering a broad range of structurally and functionally diverse proteins. Our eukaryotic cell-free translation systems are capable to provide high molecular weight membrane proteins, fluorescent-labeled membrane proteins, as well as posttranslationally modified proteins for further downstream analysis.
KW  - Cell-free protein expression
KW  - In vitro protein synthesis
KW  - Labeled membrane proteins
KW  - Synthetic glycoprotein
Y1  - 2013
U6  - https://doi.org/10.1002/elsc.201100235
SN  - 1618-0240
VL  - 13
IS  - 1
SP  - 39
EP  - 48
PB  - Wiley-Blackwell
CY  - Hoboken
ER  - 
TY  - JOUR
A1  - Broedel, A. K.
A1  - Raymond, J. A.
A1  - Duman, J. G.
A1  - Bier, Frank Fabian
A1  - Kubick, Stefan
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  -