Optimization of recombinant gene expression in Escherichia coli.
The major targets for improvement of recombinant expression efficiency in Escherichia coli are gene dosage, transcription and, to some extent, translation.  In order to evaluate the relative importance of these factors, the kinetics of specific mRNA compared to product formation was studied for different widely used expression systems, producing recombinant human superoxide dismutase. For a system employing phage T7 RNA polymerase, where a high level of recombinant protein expression puts a high metabolic burden on the cells, it was shown that transcription is not the limiting factor. To improve the translation rate of a common vector based on the tac promoter, the Shine-Dalgarno (SD) sequence was mutated towards stronger homology to the anti-SD sequence of the E. coli 16S rRNA.  A 12.2-fold increase in protein yield was accompanied by a 4.3-fold increase in specific mRNA, indicating that transcription of the recombinant gene is coupled to translation.  As this coupling amplifies the detrimental effect of a low-efficiency ribosomal binding site, much attention should be paid to translation initiation when optimizing a recombinant protein production system. Finally, reasons for the high expression level before induction are discussed, and first results towards reducing it are presented.
Mattanovich
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Institut für Angewandte Mikrobiologie, Universität für Bodenkultur, Vienna, Austria.
Weik
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Thim
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Kramer
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Bayer
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Katinger
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H
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D012321:DNA-Directed RNA Polymerases; D004794:Enzyme Repression; D004926:Escherichia coli; D005285:Fermentation; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012689:Sequence Homology, Nucleic Acid; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic; D014764:Viral Proteins
D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D014764:Viral Proteins; D013482:Superoxide Dismutase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases