TY  - JOUR
A1  - Stech, Marlitt
A1  - Merk, Helmut
A1  - Schenk, Jörg A.
A1  - Stöcklein, Walter F. M.
A1  - Wüstenhagen, Doreen Anja
A1  - Micheel, Burkhard
A1  - Duschl, Claus
A1  - Bier, Frank Fabian
A1  - Kubick, Stefan
T1  - Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system
JF  - Journal of biotechnology
N2  - Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner.
KW  - Cell-free
KW  - In vitro translation
KW  - Single chain antibody (scFv)
KW  - Insect lysate
KW  - Surface plasmon resonance
Y1  - 2012
U6  - https://doi.org/10.1016/j.jbiotec.2012.08.020
SN  - 0168-1656
VL  - 164
IS  - 2
SP  - 220
EP  - 231
PB  - Elsevier
CY  - Amsterdam
ER  - 
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  -