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  -