Use of a short A/T-rich cassette for enhanced expression of cloned genes in Escherichia coli.
A short (43-bp) A/T-rich stretch of DNA located in the intergenic region between the baiA2 and baiF genes from Eubacterium sp. strain VPI 12708 was amplified by polymerase chain reaction (PCR) and inserted in front of the Shine-Dalgarno (SD) sequences of three inefficiently-expressed Eubacterium sp. strain VPI 12708 genes cloned in Escherichia coli plasmids. Insertion of this A/T-rich cassette increased gene expression in all cases tested. Deletion of part of the A/T-rich region from a baiF clone in pUC19 resulted in decreased gene expression. Synthesis of specific mRNA was increased with addition of the A/T-rich cassette to constructs containing the baiC gene from Eubacterium sp. strain VPI 12708, but mRNA synthesis was not significantly changed in cells containing plasmid constructs with the baiF and baiG genes. Enhanced translation resulting from a decrease in mRNA secondary structure in the ribosome binding site region is discussed as a possible reason for increased gene expression with the A/T-rich cassette.
Mallonee
D H
DH
Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0678, USA.
Hylemon
P B
PB
D000595:Amino Acid Sequence; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D005051:Eubacterium / Q000235:genetics*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction
D001647:Bile Acids and Salts; D002352:Carrier Proteins; D017931:DNA Primers