. Your company has identified a novel protein that is a candidate therapeutic. H
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Question
. Your company has identified a novel protein that is a candidate therapeutic. However, analysis of cells reveals that a large fraction of the protein seems to get hung up in the ER lumen? a. What is responsible for the protein being shuttled to the ER lumen? What is the likely reason that protein accumulates within the lumen? b. In a moment of frustration, your boss wonders aloud why the protein even goes to the ER and if you could engineer the protein to skip the ER altogether. Assume you can edit the gene to remove the component that targets it to the ER, what would likely happen to the protein? c. To speed up production, your boss has suggested cloning the gene, and expressing it in vitro translation (cell free- basically a tube full of ATP, amino acids, and ribosomes, you just add the mRNA). Is this likely to result in useful (functional) protein?
Explanation / Answer
Generally proteins are exported from the cell, and are delivered to specialized organelles and retained in the closed lumen of the endoplasmic reticulum (ER) as they have to pass through the rough ER membrane.
For regulating the entry of the proteins into the cytosol, certain trafficking machinery is present. The proteins possess certain signal peptides that are recognized after their translation and the ribosome encoding that encoded these polypeptides are directed to the outer membrane of the rER to complete protein synthesis. The processed protein is placed in the lumen of the rER, or becomes embedded in it, but is not released into the cytosol.
a. What is responsible for the protein being shuttled to the ER lumen?
The protein is retained in the lumen of the ER because, it might have the ER retention signal (KDEL) at it's C-terminal end. This sequence does not allow the anchoring of the protein to the ER membrane, but it cycles back any protein detected in the Golgi.
b. In a moment of frustration, your boss wonders aloud why the protein even goes to the ER and if you could engineer the protein to skip the ER altogether. Assume you can edit the gene to remove the component that targets it to the ER, what would likely happen to the protein?
If we remove the component that targets it to the ER, the protein will not be accumulating in the ER however, it might produce a mis-folded protein.
c. An useful functional protein might not be produced in a cell free tube. As, all the required organelles, ribosomes, and enzymes would be present only in a live cell.
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