• Home
  • Search Results
  • A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates.

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

Biotechnology and bioengineering (2014-11-26)
Matthias Buntru, Simon Vogel, Katrin Stoff, Holger Spiegel, Stefan Schillberg
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sucrose, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
Potassium phosphate monobasic, ACS reagent, ≥99.0%
Sigma-Aldrich
Magnesium chloride, anhydrous, ≥98%
Sigma-Aldrich
Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M
Sigma-Aldrich
Potassium phosphate monobasic, powder, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.0%
SAFC
BIS-TRIS
Sigma-Aldrich
Sucrose, ≥99.5% (GC)
Sigma-Aldrich
Sucrose, BioXtra, ≥99.5% (GC)
Sigma-Aldrich
D-Mannitol, ≥98%
Sigma-Aldrich
DL-Dithiothreitol solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
Calcium chloride solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
Sucrose, BioUltra, for molecular biology, ≥99.5% (HPLC)
Supelco
DL-Dithiothreitol solution, 1 M in H2O
Sigma-Aldrich
Sucrose, ≥99.5%
Sigma-Aldrich
Potassium phosphate monobasic, ReagentPlus®
Supelco
Sucrose, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Sucrose, puriss., meets analytical specification of Ph. Eur., BP, NF
Sigma-Aldrich
BIS-TRIS, ≥98.0% (titration)
Sigma-Aldrich
myo-Inositol, ≥99%
Sigma-Aldrich
Nitrotetrazolium Blue chloride, ≥90.0% (HPLC)
Sigma-Aldrich
6-Benzylaminopurine, suitable for plant cell culture
Sigma-Aldrich
myo-Inositol, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture
Sigma-Aldrich
Sucrose, ACS reagent
Sigma-Aldrich
Calcium chloride, meets USP testing specifications
Sigma-Aldrich
Potassium phosphate monobasic, for molecular biology, ≥98.0%
Sigma-Aldrich
Calcium chloride, anhydrous, BioReagent, suitable for insect cell culture, suitable for plant cell culture, ≥96.0%
Sigma-Aldrich
Calcium chloride, anhydrous, powder, 99.99% trace metals basis
Sigma-Aldrich
Sucrose, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥99.5% (GC)
Supelco
Mannitol, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Magnesium chloride, powder, <200 μm