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 250g/mL of functional enhanced yellow fluorescent protein (eYFP) and about 270g/mL of firefly luciferase using plasmid templates, and up to 180g/mL eYFP using linear templates (PCR products) in 18h 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 (approximate to 150g/mL), the model enzyme glucose oxidase (GOx) (approximate to 7.3U/mL), and a transmembrane growth factor (approximate to 25g/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. (c) 2014 Wiley Periodicals, Inc.