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Use of a short A/T-rich cassette for enhanced expression of cloned genes inEscherichia coli

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Abstract

A short (43-bp) A/T-rich stretch of DNA located in The intergenic region between thebaiA2 andbaiF genes fromEubacterium sp. strain VPI 12708 was amplified by polymerase chain reaction (PCR) and inserted in front of the Shine-Dalgarno (SD) sequences of three inefficiently-expressedEubacterium sp. strain VPI 12708 genes cloned inEschcrichia 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 abaiF 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 thebaiC gene from Eubacterium sp. strain VPI 12708, but mRNA synthesis was not significantly changed in cells containing plasmid constructs with thebaiF andbaiG 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.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, PO Box 980678, 23298-0678, Richmond, VA

    Darrell H. Mallonee & Phillip B. Hylemon

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  1. Darrell H. Mallonee
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  2. Phillip B. Hylemon
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Mallonee, D.H., Hylemon, P.B. Use of a short A/T-rich cassette for enhanced expression of cloned genes inEscherichia coli . Mol Biotechnol 11, 27–35 (1999). https://doi.org/10.1007/BF02789174

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