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

 
: Buntru, M.; Vogel, S.; Stoff, K.; Spiegel, H.; Schillberg, S.

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Biotechnology & bioengineering 112 (2015), No.5, pp.867-878
ISSN: 0006-3592
ISSN: 1097-0290
English
Journal Article
Fraunhofer IME ()

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 μg/mL of functional enhanced yellow fluorescent protein (eYFP) and about 270 μg/mL of firefly luciferase using plasmid templates, and up to 180 μg/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 μg/mL), the model enzyme glucose oxidase (GOx) (∼7.3 U/mL), and a transmembrane growth factor (∼25 μg/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.

: http://publica.fraunhofer.de/documents/N-356181.html