English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT

Released

Journal Article

Troubleshooting coupled in in vitro transcription–translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins

MPS-Authors

Iskakova,  Madina B.
Max Planck Society;

/persons/resource/persons50510

Szaflarski,  Witold
Ribosomes, Max Planck Institute for Molecular Genetics, Max Planck Society;

Dreyfus,  Marc
Max Planck Society;

/persons/resource/persons50444

Nierhaus,  Knud
Ribosomes, Max Planck Institute for Molecular Genetics, Max Planck Society;

External Resource
No external resources are shared
Fulltext (restricted access)
There are currently no full texts shared for your IP range.
Fulltext (public)

e135.pdf
(Any fulltext), 2MB

Supplementary Material (public)
There is no public supplementary material available
Citation

Iskakova, M. B., Szaflarski, W., Dreyfus, M., Remme, J., & Nierhaus, K. (2006). Troubleshooting coupled in in vitro transcription–translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins. Nucleic Acids Research, 34(19), e135-e135. doi:10.1093/nar/gkl462.


Cite as: https://hdl.handle.net/11858/00-001M-0000-0010-8416-E
Abstract
Cell-free coupled transcription–translation systems with bacterial lysates are widely used to synthesize recombinant proteins in amounts of several mg per ml. By using reporter green fluorescence protein (GFP) we demonstrate that proteins are synthesized with an unsatisfyingly low-active fraction of (50 ± 20)%. One reason is probably the T7 polymerase used, being up to eight times faster than the intrinsic transcriptase and thus breaking the coupling between transcription and translation in bacterial systems. The active fraction of the synthesized protein was improved by using either a slower T7 transcriptase mutant or lowering the incubation temperature to 20°C. A drop of protein synthesis observed after 7 h incubation time was not due to a shortage of nucleotide triphosphates, but rather to a shortage of amino acids. Accordingly, a second addition of amino acids after 10 h during an incubation at 20°C led to synthesis of up to 4 mg/ml of GFP with virtually 100% activity.