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

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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. Biotechnol. Bioeng. 2015;112: 867–878.

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