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Nucleic Acids Research, 1990, Vol. 18, No. 7 1711-1718
© 1990

MOLECULAR BIOLOGY

The use of two-cistron constructions in improving the expression of a heterologous gene in E.coli

A.J. Makoff and A.E. Smallwood

Department of Molecular Biology, Wellcome Biotech, Langley Court Beckenham, Kent BR3 3BS, UK

* To whom correspondence should be addressed

Received January 30, 1990. Accepted March 2, 1990.

Many heterologous genes when cloned into bacterial expression vectors are poorly expressed because of an inefficient ribosome binding site (RBS). We have constructed a plasmid which expresses human {gamma}-interferon ({gamma}-IF), where the level of expression is limited by the RBS. Expression was increased by placing the {gamma}-IF sequence immediately downstream of a small translated sequence. The production of {gamma}-IF was dependent upon the efficiency of translation of this upstream cistron and could be increased to very high levels. The same upstream cistron would greatly improve the expression of {gamma}-IF in a plasmid where the RBS was very poor due to inhibitory secondary structure at the 5' end of its mRNA. However, it would not improve the efficiency of a poor RBS containing a weak Shine-Dalgarno sequence. The general utility of the two-cistron expression strategy to diagnose a weak RBS is discussed.


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S. Kimura, T. Umemura, and T. Iyanagi
Two-Cistronic Expression Plasmids for High-Level Gene Expression in Escherichia coli Preventing Translational Initiation Inhibition Caused by the Intramolecular Local Secondary Structure of mRNA
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