You are working on a protein for a research project. The protein does not express well in prokaryotic expression systems so you decide to try a eukaryotic system, baker’s yeast, instead. As part of the expression system, you add a signal peptide to the protein so that it will be exported through the endomembrane system to the outside of the yeast cell. This makes purifying the protein much simpler. During the purification, you notice that the protein is heavier than it should be during gel filtration chromatography. This is confirmed by SDS-PAGE. What could be the reason that the protein has gained mass when expressed in the eukaryotic system?

You are working on a protein for a research project. The protein does not express well in prokaryotic expression systems so you decide to try a eukaryotic system, baker’s yeast, instead. As part of the expression system, you add a signal peptide to the protein so that it will be exported through the endomembrane system to the outside of the yeast cell. This makes purifying the protein much simpler. During the purification, you notice that the protein is heavier than it should be during gel filtration chromatography. This is confirmed by SDS-PAGE. What could be the reason that the protein has gained mass when expressed in the eukaryotic system?

Biology Today and Tomorrow without Physiology (MindTap Course List)

5th Edition

ISBN:9781305117396

Author:Cecie Starr, Christine Evers, Lisa Starr

Publisher:Cecie Starr, Christine Evers, Lisa Starr

Chapter7: Gene Expression And Control

Section: Chapter Questions

Problem 4CT

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Imagine that you and your colleagues are working in a lab to develop a protein synthesis system for a new type of synthetic cell. During your brainstorming sessions, you propose that polycistronic mRNA would be much more useful than mRNA that is only translated into one protein. This would allow for multiple proteins necessary for a particular function to be translated together. One of your colleagues says that is a good idea, but if you decide to go with polycistronic mRNA, you’d better make sure to use a prokaryotic translation system. Why might it be problematic to use a eukaryotic translation system with polycistronic mRNA? Mention three reasons:
You are a research scientist working in genetic engineering. You create a piece of DNA that you want to express in E. coli, a prokaryote. This piece of DNA (represented by the schematic in the figure) consists of a bacterial promoter, a ribosome binding sequence (RBS), a eukaryotic gene and a terminator sequence. Do you think that this piece of DNA would be expressed if placed into an E. coli cell that contains all the machinery needed for gene expression? Fully motivate your answer.
Shown below is a schematic drawing of a gene, with the transcription unit divided into numbered regions. The arrows (;) indicate transcription initiation sites, "D" indicates a splice donor site, "A" indicates a splice acceptor site, and "An" indicates a polyadenylation signal. Give all the possible fully processed mRNAs that could be produced from transcripts of this gene (you don't need to draw anything, just list the regions that would be included in each mRNA by number).
Describes a method known as Western blotting that can be used to detect a polypeptide that is translated from a particular mRNA. In this method, a particular polypeptide or protein is detected by an antibody that specifically recognizes a segment of its amino acid sequence. After the antibody binds to the polypeptide within a gel, a secondary antibody (which is labeled) is used to visualize the polypeptide as a dark band.For example, an antibody that recognizes α-galactosidase A couldbe used to specifically detect the amount of α-galactosidase A proteinon a gel. The enzyme α-galactosidase A is defective in individuals with Fabry disease, which shows an X-linked recessive pattern of inheritance. Amy, Nan, and Pete are siblings, and Pete has Fabry disease. Aileen, Jason, and Jerry are brothers and sister, and Jerry has Fabry disease. Amy, Nan, and Pete are not related to Aileen, Jason, and Jerry. Amy, Nan, and Aileen are concerned that they could be carriers of a defective…
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You are a research scientist working in genetic engineering. You create a piece of DNA that you want to express in E. coli, a prokaryote. This piece of DNA consists of a bacterial promoter, a ribosome binding sequence (RBS), a eukaryotic gene and a terminator sequence. Do you think that this piece of DNA would be expressed if placed into an E. coli cell that contains all the machinery needed for gene expression?
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You are interested in a eukaryotic protein involved in immunity, and you are attempting to express this protein in E. coli in order to produce large amounts of the protein. You take your DNA construct and place it into E. coli. You measure protein expression and notice that no protein is being made. Provide 1 reason why you don't see protein as it relates to transcription and how this problem could be resolved.