A yeast intron as a translational terminator in a plasmid shuttle vector.
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.
Gibbs
Moreland D
MD
Biotechnology Research Institute and Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia.
Reeves
Rosalind A
RA
Sunna
Anwar
A
Bergquist
Peter L
PL
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D007438:Introns / Q000235:genetics*; D007716:Kluyveromyces / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D014176:Protein Biosynthesis; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013728:Terminator Regions, Genetic / Q000235:genetics*
D011994:Recombinant Proteins