Manipulating gene expression & protein production 1) Inducible promoters a) What

Question

Manipulating gene expression & protein production 1) Inducible promoters a) What is a strong promoter, and why is it desirable? b) Why is constitutive expression problematic? 2) T7 promoter system a) What is the advantage of a viral T7 RNA polymerase? b) What are the two points where addition of lactose turns on gene expression? 3) Mammalian expression a) b) What is the difference between transient and stable cell lines? Why is it beneficial to use cancerous cells in protein production? 4) Purification a) Chromatography i) What properties do the following resins separate proteins by: (1) Affinity (2) lon exchange (3) Size exclusion How can fusion proteins help in purification: i) His-tag? i MBP-tag? b) c) Protein buffers How is buffer exchanged by: (1) Dialysis? (2) Gel filtration columns? i)

Explanation / Answer

a. RNA polymerase binds more tightly on strong promoter with high affinity and leads to more transcripts.

b. Constitutive expression means continuous expression. In biological system all proteins are synthesized and sectreted whenever there need occurs. Constitutive exprssion of genes does not make proteins according to demand and leads to abnormalities and disease.

2)

a. It have more turn over capasity. More productive polymerase.

b. lactose converts into allolactose which inhibits the activity of repressor and turn on the gene expression.

Lactose makes the protein permease by gene expresion which allows more incoming of lactose into the cell by permease channel.

I answered according to chegg guidlined. for more answers upload each questions separately.

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