A yeast intron as a translational terminator in a plasmid shuttle vector

Moreland D. Gibbs, Rosalind A. Reeves, Anwar Sunna, Peter L. Bergquist

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Plasmid shuttle vectors that contain both prokaryotic (Escherichia coli) and eukaryotic origins of replication are routinely used in molecular biology since E. coli is generally the organism of choice for manipulation of recombinant DNA. Initial transformation of the shuttle vector into E. coli allows production of microgram quantities of DNA suitable for transformation of low-transformation-efficiency hosts. A shuttle/expression vector for the yeast Kluyveromyces lactis, pCWK1, allows recombinant protein fused to the killer toxin signal sequence to be secreted to the medium. The heterologous genes are transcribed under the control of the K. lactis LAC4 promoter, which is tightly regulated in K. lactis. However, in E. coli the LAC4 promoter functions constitutively, and as a result, uncontrolled transcription and translation of genes that are toxic in E. coli can result in cell death, and subsequent failure to recover intact E. coli transformants. We have constructed and tested a modified shuttle vector that contains a K. lactis ribosomal intron that acts as a translational terminator in E. coli, preventing or reducing the expression of recombinant proteins and avoiding toxicity. When transcribed in K. lactis, the intron is spliced from the mRNA allowing the translation of intact full-length, active recombinant gene product.

LanguageEnglish
Pages573-577
Number of pages5
JournalFEMS Yeast Research
Volume4
Issue number6
DOIs
Publication statusPublished - Mar 2004

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Genetic Vectors
Introns
Plasmids
Yeasts
Escherichia coli
Recombinant Proteins
Genes
Kluyveromyces
Replication Origin
Recombinant DNA
Poisons
Protein Biosynthesis
Protein Sorting Signals
Molecular Biology
Cell Death
DNA

Cite this

Gibbs, Moreland D. ; Reeves, Rosalind A. ; Sunna, Anwar ; Bergquist, Peter L. / A yeast intron as a translational terminator in a plasmid shuttle vector. In: FEMS Yeast Research. 2004 ; Vol. 4, No. 6. pp. 573-577.
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A yeast intron as a translational terminator in a plasmid shuttle vector. / Gibbs, Moreland D.; Reeves, Rosalind A.; Sunna, Anwar; Bergquist, Peter L.

In: FEMS Yeast Research, Vol. 4, No. 6, 03.2004, p. 573-577.

Research output: Contribution to journalArticleResearchpeer-review

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