Troubleshooting coupled in vitro transcription-translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins.
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 degrees 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 degrees C led to synthesis of up to 4 mg/ml of GFP with virtually 100% activity.
Iskakova
Madina B
MB
Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestrasse 73, D-14195 Berlin, Germany.
Szaflarski
Witold
W
Dreyfus
Marc
M
Remme
Jaanus
J
Nierhaus
Knud H
KH
D002474:Cell-Free System; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
D012333:RNA, Messenger; D011994:Recombinant Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases