Question

A group of investigators has cloned a gene encoding a mouse enzyme and attempted to express it in yeast. The gene is functional in yeast but expressed at very low levels. Based on your understanding of transcription and translation answer the following questions.

1)What could account for the low expression levels seen in the yeast?

2}How might the investigators alter their clone to achieve higher expression levels in yeast without altering the protein coding region

Answer 1

Answer 1:)

Yeast is a plant expression system and hence mammalian expression may get difficult in yeast. The main reason is Plasmid intability in yeast. That is because the use of hybrid plasmids, which are often use in yeast to introduce foreign DNA.

Answer 2:)

To overcome this problem, we need to use immobilize system for the growth of the cells. Moreover, if medium is non-selective, then we will also get more expression.

Yes, we obtained the PCR product. The size of the product is 200bp.

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Answer 2

1. There could be several reasons for the low expression levels of the mouse enzyme gene in yeast. One possibility is that the promoter region of the gene may not be compatible with the yeast transcriptional machinery, leading to inefficient initiation of transcription. Additionally, post-transcriptional and post-translational modifications that are necessary for optimal expression and activity of the enzyme in mouse cells may not be present or functional in yeast. Furthermore, the codon usage bias between mouse and yeast may also contribute to low expression levels, as the frequency of optimal codons for the yeast translation machinery may be lower in the mouse gene.

2. To achieve higher expression levels of the mouse enzyme gene in yeast, the investigators could alter various regulatory elements of the gene. For instance, they could replace the original promoter region with a strong promoter that is compatible with the yeast transcriptional machinery, such as the yeast alcohol dehydrogenase (ADH) promoter. They could also introduce enhancer sequences to further boost transcriptional activity. Additionally, they could modify the 5' and 3' untranslated regions (UTRs) of the mRNA to optimize translational efficiency and stability in yeast. Other modifications such as altering the copy number of the gene or optimizing the culture conditions for growth and expression may also be considered. It is important to note that these alterations should not affect the protein coding region of the gene to ensure that the expressed enzyme retains its original activity and specificity.