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

AUTOR(ES)

Makoff, A J

RESUMO

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.

Documentos Relacionados

• Translation of a synthetic two-cistron mRNA in Escherichia coli.
• In vivo gene expression directed by synthetic promoter constructions restricted to the -10 and -35 consensus hexamers of E. coli.
• Expression of polyoma early gene products in E. coli.
• Cloning and expression of a porcine prorelaxin gene in E. coli.
• Direct expression of hepatitis B surface antigen gene in E. coli.