Open AccessThe use of two-cistron constructions in improving the expression of a heterologous gene inE.coli
Author(s) -
Andrew Makoff,
Althea E. Smallwood
Publication year - 1990
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/18.7.1711
Subject(s) - cistron , biology , ribosomal binding site , heterologous , plasmid , gene , genetics , microbiology and biotechnology , gene expression , sequence (biology) , messenger rna , heterologous expression , translation (biology) , rna , recombinant dna
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.