Hi everyone!!
I never have worked with S. cervisiae and need to permeabilise for IFI... Is it possible or I need to induce spheroplast?
Thanks...
These two references will provide the methods to permeabilize S cereivise
http://www.ncbi.nlm.nih.gov/pubmed/20890900
http://www.ncbi.nlm.nih.gov/pubmed/15058989
Curr Protoc Mol Biol. 2010 Oct;Chapter 13:Unit 13.2B. doi: 10.1002/0471142727.mb1302bs92.
Drug-induced permeabilize of S. cerevisiae.
Budding yeast are not permeable to many drugs. This unit provides a protocol in which polygodial is used to permeabilize the cell membrane, thereby allowing budding yeast cells to be treated with drugs that otherwise would be ineffective.
© 2010 by John Wiley & Sons, Inc.
Biotechnol Prog. 2004 Mar-Apr;20(2):449-56.
Enzyme encapsulation in permeabilized Saccharomyces cerevisiae cells.
Chow CK1, Palecek SP.
The Saccharomyces cerevisiae cell wall provides a semipermeable barrier that can retain intracellular proteins but still permits small molecules to pass through. When S. cerevisiae cells expressing E. coli lacZ are treated with detergent to extract the cell membrane, beta-galactosidase activity in the permeabilized cells is approximately 40% of the activity of the protein in cell extract. However, the permeabilized cells can easily be collected and reused over 15 times without appreciable loss in activity. Cell wall composition and thickness can be modified using different cell strains for enzyme expression or by mutating genes involved in cell wall biosynthesis or degradation. The Sigma1278b strain cell wall is less permeable than the walls of BY4742 and W303 cells, and deleting EXG1, which encodes a 1,3-beta-glucanase, can further reduce permeability. A short Zymolyase treatment can increase cell wall permeability without rupturing the cells. Encapsulating multiple enzymes in permeabilized cells can offer kinetic advantages over the same enzymes in solution. Regeneration of ATP from AMP by adenylate kinase and pyruvate kinase encapsulated in the same cell proceeded more rapidly than regeneration using a cell extract. Combining permeabilized cells containing adenylate kinase with permeabilized cells containing pyruvate kinase can also regenerate ATP from AMP, but the kinetics of this reaction are slower than regeneration using cell extract or permeabilized cells expressing both enzymes.
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