You want to clone a eukaryotic gene and express the corresponding protein in yeast. However, the protein typically localizes within mitochondria
How will you perform your gene cloning so that the protein is secreted from the cell, rather than localized within yeast mitochondria?
What will be an ideal response?
Answer: The cloned gene will need to lack the signal sequences associated with localization within mitochondria. The first segment of the cloned gene (coding for the N-terminus of the protein) should contain an ER signal sequence that directs the ribosome-mRNA-polypeptide complex to the surface of the rough endoplasmic reticulum. This portion of the gene will need to be removed; the mRNA resulting from the engineered gene should then be translated on cytoplasmic ribosomes. As long as the protein is soluble, it should then follow the default pathway from the ER lumen, to the Golgi apparatus, and finally to secretory vesicles that move to the cell surface and fuse with the plasma membrane.
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