High-level synthesis in Escherichia coli of the SV40 small-t antigen under control of the bacteriophage lambda pL promoter |
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Authors: | C Derom D Gheysen W Fiers |
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Affiliation: | Laboratory of Molecular Biology, State University of Ghent, Ledeganckstraat 35, B-9000 GhentBelgium |
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Abstract: | Several plasmids were constructed in which the SV40 small-t antigen gene was inserted in close proximity downstream from the thermoinducible leftward promoter (pL) of bacteriophage lambda. Upon temperature induction the best of our constructions expressed a small-t-related 19 000-dalton polypeptide in an amount corresponding to approx. 2.5% of total de novo protein synthesis. This 19 000-dalton protein was identified as small-t by specific immunoprecipitation with anti-T serum and by two-dimensional fingerprint analysis. In addition to the 19 000-dalton product, representative plasmids expressed fairly large amounts (up to 7% of total de novo protein synthesis) of a protein with an apparent Mr of 14 500. This 14 500-dalton polypeptide was shown to be related to authentic small-t. Presumably the secondary structure of the mRNA starting at pL is such that translation initiation at an internal AUG codon of the small-t gene is favored over initiation at the true initiating codon. |
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Keywords: | Recombinant DNA exonuclease treatment tryptic peptide analysis internal translation initiation K kilodaltons SD Shine and Dalgarno (1975) sequence SDS sodium dodecyl sulfate SV40 simian virus 40 |
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