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 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.
authors.affiliation: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, Aachen, 52074, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Buntru
authors.forename: Simon
authors.initials: S
authors.lastname: Vogel
authors.forename: Katrin
authors.initials: K
authors.lastname: Stoff
authors.forename: Holger
authors.initials: H
authors.lastname: Spiegel
authors.forename: Stefan
authors.initials: S
authors.lastname: Schillberg
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001709:Biotechnology / Q000379:methods; D002474:Cell-Free System / Q000378:metabolism*; D002478:Cells, Cultured; D006031:Glycosylation; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology* / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D049409:Luciferases, Firefly
keywords: BY-2 cell lysate; cell-free protein synthesis; coupled transcription-translation; high-throughput screening; protein expression