Functional expression of the cystic fibrosis transmembrane conductance regulator in yeast
| Autor: | John Cuppoletti, Sanjoy K. Mahanty, Pingbo Huang, Katarina Stroffekova, Gene A. Scarborough |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
congenital
hereditary and neonatal diseases and abnormalities ATPase Saccharomyces cerevisiae Biophysics Gene Expression Chloride ion channel Expression Sf9 Spodoptera Biochemistry law.invention law Centrifugation Density Gradient Animals Humans Initiation codons biology Chemistry Vesicle Cell Membrane Cell Biology respiratory system biology.organism_classification Cyclic AMP-Dependent Protein Kinases Recombinant Proteins Yeast Cystic fibrosis transmembrane conductance regulator respiratory tract diseases Cell biology Membrane Mutagenesis Site-Directed Recombinant DNA biology.protein Plasmids |
| Zdroj: | Hong Kong University of Science and Technology |
| ISSN: | 0005-2736 |
| Popis: | Recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) has been produced in a Saccharomyces cerevisiae expression system used previously to produce transport ATPases with high yields. The arrangement of the bases in the region immediately upstream from the ATG start codon of the CFTR is extremely important for high expression levels. The maximal CFTR expression level is about 5–10% of that in Sf9 insect cells as judged by comparison of immunoblots. Upon sucrose gradient centrifugation, the majority of the CFTR is found in a light vesicle fraction separated from the yeast plasma membrane in a heavier fraction. It thus appears that most of expressed CFTR is not directed to the plasma membrane in this system. CFTR expressed in yeast has the same mobility (ca. 140 kDa) as recombinant CFTR produced in Sf9 cells in a high resolution SDS-PAGE gel before and after N-glycosidase F treatment, suggesting that it is not glycosylated. The channel function of the expressed CFTR was measured by an isotope flux assay in isolated yeast membrane vesicles and single channel recording following reconstitution into planar lipid bilayers. In the isotope flux assay, protein kinase A (PKA) increased the rate of 125I− uptake by about 30% in membrane vesicles containing the CFTR, but not in control membranes. The single channel recordings showed that a PKA-activated small conductance anion channel (8 pS) with a linear I–V relationship was present in the CFTR membranes, but not in control membranes. These results show that the human CFTR has been expressed in functional form in yeast. With the reasonably high yield and the ability to grow massive quantities of yeast at low cost, this CFTR expression system may provide a valuable new source of starting material for purification of large quantities of the CFTR for biochemical studies. |
| Databáze: | OpenAIRE |
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