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  4. A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates
 
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2015
Journal Article
Title

A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates

Abstract
Cell-free protein synthesis is a powerful method for the high-throughput production of recombinant proteins, especially proteins that are difficult to express in living cells. Here we describe a coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates (BYLs). Using a combination of fractional factorial designs and response surface models, we developed a cap-independent system that produces more than 250 mg/mL of functional enhanced yellow fluorescent protein (eYFP) and about 270 mg/mL of firefly luciferase using plasmid templates, and up to 180 mg/mL eYFP using linear templates (PCR products) in 18 h batch reactions. The BYL contains actively-translocating microsomal vesicles derived from the endoplasmic reticulum, promoting the formation of disulfide bonds, glycosylation and the cotranslational integration of membrane proteins. This was demonstrated by expressing a functional full-size antibody (∼150 mg/mL), the model enzyme glucose oxidase (GOx) (∼7.3 U/mL), and a transmembrane growth factor (∼25 mg/mL). Subsequent in vitro treatment of GOx with peptide-N-glycosidase F confirmed the presence of N-glycans. Our results show that the BYL can be used as a high-throughput expression and screening platform that is particularly suitable for complex and cytotoxic proteins.
Author(s)
Buntru, M.
Vogel, S.
Stoff, K.
Spiegel, H.
Schillberg, S.
Journal
Biotechnology & bioengineering  
DOI
10.1002/bit.25502
Language
English
Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie IME  
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