Question

Imagine this story. After talking about the highly specific TEV protease (its substrate is the peptide sequence ENLYFQS) in class I noticed that the HIV polymerase has along its surface the sequence ENLYYQS, which varies at only one position from the TEV substrate, the substitution of an aromatic Y for an aromatic F (not actually true). A survey of the literature showed that this sequence is not cleaved by TEV. I thought if we could isolate a variant (mutated) TEV enzyme that could cleave ENLYYQS, then we could insert the gene for this new enzyme into an AIDS patient’s hematopoietic stem cells and return these cells to the patients to take over the T-cell population with cells that are resistant to HIV because the pol protein would be disrupted. The first step is to isolate a variant that would cleave the ENLYYQS target sequence intracellularly. Inspired by Virginia Cornish’s 3-hybrid approach to isolate an improved cellulase, I would like to randomly mutagenize the active site of TEV protease and select from a pool of such mutants a mutant TEV gene that codes for the most active enzyme in this regard. To be sure that the enzyme would work in mammalian cell, I want to use human HEK293 cells as hosts.

Describe an experimental plan for this idea. Include:
The mutagenesis, the bait, the prey and the third hybrid component. You may assume the yeast gal DNA binding components work in mammalian cells, but not the yeast activator domain (come up with a mammalian alternative). Another problem is that there are no URA-3 mutants for HEK293 cells, or any mutants you can use, so come up some alternative strategy. Be sure to include a clearly labeled diagram to explain your plan. And be sure to include all elements required for mammalian gene expression in any gene constructs you create.

Here’s an outline to help you plan:

1. A DNA binding domain of a DNA binding protein. You choose: ___________________

2. A stretch of DNA to which the binding domain will bind (name it): _________ ___________

3. An activation domain known to be active in mammalian cells: ______________________

4. A reporter gene to select cells you want _______________________________

5. Any additions to the medium components that may be required ___________________

Fill in your choices for the numbered blanks above and then explain them below. You may discuss your choices with your classmates but your explanation should be in your own words. Then explain how your system would function. Be sure to include a diagram and the selection conditions.

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Random mutagenesis of TEVprotease:
For random mutagenesis of TEV protease we will use Genemorph mutagenesis kit from Aigilent Technologies.
This is a PCR based kit that uses the enzyme Mutazyme II DNA polymerase that has low fidelity.
We will take TEV protease CDNA cloned in any cloning vector such as PSKII (+) and perform a PCR as specified in the kit.
Following this, we will clone the PCR product in any expression vector, such as PCDNA3.1 preferably with a tag such as a YFP tag (in frame with TEV polymerase reading frame). Thus we obtain a library of randomly mutagenized TEV protease in an expression vector. (The tag YFP will help us in monitoring the transfection efficiency during the subsequent experiments....

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