The use of two-cistron constructions in improving the expression of a heterologous gene in E. 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 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.
Makoff
A J
AJ
Department of Molecular Biology, Wellcome Biotech, Beckenham, Kent, UK.
Smallwood
A E
AE
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005796:Genes; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D012333:RNA, Messenger; D011994:Recombinant Proteins; D007371:Interferon-gamma