Gibbs Moreland D, Reeves Rosalind A, Sunna Anwar, Bergquist Peter L
Biotechnology Research Institute and Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia.
FEMS Yeast Res. 2004 Mar;4(6):573-7. doi: 10.1016/j.femsyr.2003.11.001.
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.
由于大肠杆菌通常是用于操作重组DNA的首选生物体，因此含有原核（大肠杆菌）和真核复制起点的质粒穿梭载体在分子生物学中经常被使用。将穿梭载体最初转化到大肠杆菌中可产生微克量的DNA，这些DNA适合用于转化低转化效率的宿主。一种用于乳酸克鲁维酵母的穿梭/表达载体pCWK1，可使与杀伤毒素信号序列融合的重组蛋白分泌到培养基中。异源基因在乳酸克鲁维酵母LAC4启动子的控制下转录，该启动子在乳酸克鲁维酵母中受到严格调控。然而，在大肠杆菌中，LAC4启动子组成型发挥作用，结果，对大肠杆菌有毒性的基因的不受控制的转录和翻译会导致细胞死亡，并随后无法获得完整的大肠杆菌转化体。我们构建并测试了一种改良的穿梭载体，该载体含有一个乳酸克鲁维酵母核糖体内含子，该内含子在大肠杆菌中作为翻译终止子，可防止或减少重组蛋白的表达并避免毒性。当在乳酸克鲁维酵母中转录时，内含子从mRNA中剪接出来，从而允许完整全长活性重组基因产物的翻译。
