In vivo overexpression and purification ofEscherichia colitRNAser
| Autor: | Michael Härtlein, Franck Borel, Reuben Leberman |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Overexpression
Molecular Sequence Data Biophysics Biology Molecular cloning medicine.disease_cause Biochemistry chemistry.chemical_compound Plasmid Structural Biology Serine Escherichia coli Genetics medicine tRNA Molecular Biology RNA Transfer Ser Base Sequence Aminoacyl tRNA synthetase RNA Cell Biology Molecular biology Terminator (genetics) chemistry Genes Bacterial Transfer RNA Aminoacyl-tRNA synthetase Low copy number Plasmids |
| Zdroj: | FEBS Letters. 324:162-166 |
| ISSN: | 0014-5793 |
| DOI: | 10.1016/0014-5793(93)81385-d |
| Popis: | DNA fragments corresponding to the sequences of Escherichia coli tRNA(2ser) and amber suppressor tRNA(ser), were synthesized from overlapping oligonucleotides. These were interposed between a strong promotor and a synthetic transcriptional terminator to ensure the production of a transcript of the correct size. The genes of promotor, fragment and terminator were cloned into a conditional runaway replication plasmid. At temperatures below 37 degrees C this vector has a low copy number but, following a temperature shift to 42 degrees C, the copy number is no longer regulated. Using these constructs an overexpression of tRNA(ser) of about 20 times the level of the wild-type pool could be obtained (corresponding e.g. to 200 times the expression tRNA(2ser)). From these systems 10 mg quantities of tRNA(ser)s could be isolated with a serine acceptance of 1,100 pmol/A280 unit. |
| Databáze: | OpenAIRE |
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