Use of a portable ribosome-binding site for maximizing expression of a eukaryotic gene in Escherichia coli |
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| Authors: | D M Marquis J M Smolec D H Katz |
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| Affiliation: | 1. Department of Cardiology and Institute of Vascular Medicine, Peking University Third Hospital, Beijing 100191, China;2. Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China;3. State Key Laboratory of Membrane Biology, College of Life Sciences, Peking University, Beijing 100871, China;4. Department of Biomedical Informatics, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Science of the Ministry of Education Center for Non-coding RNA Medicine, Peking University, Beijing 100191, China;5. State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China;6. NHC Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides, Beijing 100191, China;7. Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing 100191, China;8. Beijing Key Laboratory of Cardiovascular Receptors Research, Beijing 100191, China |
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| Abstract: | To maximize expression of a eukaryotic gene in Escherichia coli, a series of plasmids were constructed containing various synthetic ribosome-binding sites (RBS). These sites consist of a Shine-Dalgarno (SD) region (with translation stop codons in all three reading frames) positioned at distances 5-9 nucleotides (nt) from the AUG initiator codon of the gene coding for human T-cell growth factor (TCGF or IL-2). The region encompassing the RBS through the TCGF structural gene from each of these plasmids was inserted as a 'cassette' into seven different E. coli expression vectors, and TCGF production was measured. Our results demonstrate a greater than 2000-fold range of TCGF synthesis dependent upon the promoter and the synthetic RBS used. The translational efficiency of the TCGF gene was found to be influenced by the quality of the RBS, which is in part determined by the external sequence context of this site. The synthetic RBS, containing the necessary information for the translation initiation process, readily accessible by restriction sites, should be of general usefulness in obtaining maximum expression of eukaryotic genes in E. coli. |
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