THE USE OF 2-CISTRON CONSTRUCTIONS IN IMPROVING THE EXPRESSION OF A HETEROLOGOUS GENE IN ESCHERICHIA-COLI

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 γ-interferon (γ-IF), where the level of expression is limited by the RBS. Expression was increased by placing the γ-IF sequence immediately downstream of a smalltranslated sequence. The production of γ-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 γ-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. © 1990 Oxford University Press.