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Related Experiment Videos

A polylinker-derived sequence, PL, highly increased translation efficiency in Escherichia coli.

J Xu1, R Mironova, I G Ivanov

  • 1Department of Botany, University of Toronto, Ontario, Canada. jxu@cbr.med.harvard.edu

Journal of Basic Microbiology
|March 11, 1999
PubMed
Summary

A novel polylinker-derived sequence (PL) enhances E. coli protein production by over 2-fold compared to the Shine-Dalgarno (SD) sequence, facilitating high-level gene expression.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • Pokeweed antiviral protein (PAP) is a potent ribosome-inactivating glycosidase.
  • Standard gene expression in E. coli relies on the Shine-Dalgarno (SD) sequence for translation initiation.

Purpose of the Study:

  • To investigate the high expression of PAP in E. coli lacking a canonical SD sequence.
  • To identify and characterize the translational initiation mechanism of the PAP gene.
  • To evaluate the potential of the identified sequence for broad application in heterologous gene expression.

Main Methods:

  • Cloning the PAP gene into an expression vector with and without a polylinker-derived sequence (PL).
  • Comparing protein yields in E. coli transformed with vectors containing PL versus a consensus SD sequence.

Related Experiment Videos

  • Mutagenesis of identified 5' terminal sequences in PAP mRNA and assessment of their impact on protein yield.
  • Utilizing reporter genes (human calcitonin) to assess PL's efficacy in expressing other genes.

    • Main Results:

    • E. coli produced over twice the amount of PAP using a PL sequence compared to a consensus SD sequence.
    • Computer analysis revealed two rRNA-complementary sequences (box I and box II) in the PAP mRNA 5' region.
    • Mutation of box I or box II significantly decreased PAP yield, indicating their essential role in translation.
    • The PL sequence doubled the expression level of human calcitonin genes compared to SD.

    Conclusions:

    • The PL sequence acts as an efficient translational initiator in E. coli.
    • The PAP mRNA possesses intrinsic translation initiation elements that enhance gene expression.
    • The PL sequence offers a promising strategy for achieving high-level expression of specific genes in E. coli.