BeHappy Receptor Overexpression
You have just joined a new lab and you are about to start a new and stimulating project.

The BeHappy receptor (BHR1) that controls the euphoric cascade (that is involved in most drug abuse phenomena) has recently been cloned in your new lab.

     You have (only) two assignments: 

A. Establish an expression system, that will allow further characterization of the receptor's mode of operation and the development of new drugs that will enhance euphoric reaction mediated by the BHR1-induced cascade. 

B. Express BHR1 in high amounts  for developing in-vitro assays.
Continue
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

The known characteristics of BHR1 are as follows:

1. BHR1 was initially cloned from human brain cDNA library. It is uniquely expressed in neuronal tissue.
2. BHR1 is located in the plasma membrane.
3. Glycosylation of the protein is essential for proper folding and function of BHR1.
4. The receptor is a homotetramer.
5. The MW of each subunit is 40Kd.
6. When studied in neuronal cultures, BHR1 was present in trace amounts. Attempts of over expressing the receptor resulted in neuronal cell death.

Based on the above mentioned characteristics, what features should your expression system support?
(Examine ALL answers. Choose between right and wrong.)

1. The expression system should allow secretion of the protein
2. The expression system should support eukaryotic post-translational modifications
3. The expression vector should express the four genes encoding the four subunits
4. The receptor seems to be toxic to neuronal cells, therefore it will be beneficial to express it in mice fibroblasts
5. The receptor seems to be toxic to neuronal cells, therefore it will be beneficial to express it in an inducible system
6. The receptor seems to be toxic to neuronal cells, therefore it will be beneficial to express it in bacterial cells
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You chose to focus on the characterization of the receptor mode of action (assignment A).
Based on the  requirements defined in the previous question, which expression system should you choose (Check all answers. Choose between wrong and possible.)
1. T7-promoter induced vectors for bacterial expression system
2. Induced stable expression in insect cells
3. Baculovirus expression system
4. Induced stable expression in MDCK cells from kidney (These cells are well-characterized, easy to manipulate and grow well in cell culture)
5. Induced stable expression in neuronal cells
6. Viral mediated expression system for mammalian cells. (i.e. Adenovirus, vaccinia virus etc.)

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You have chosen to establish an overexpression system for in-vitro assay. (assignment B).
Based on the above requirements which expression system should you choose 
(Check all answers. Choose between wrong and possible.)
1. T7-promoter induced vectors for bacterial expression system
2. Expression in yeast cells.
3. Baculovirus expression system
4. Induced stable expression in MDCK cells from kidney (These cells are well-characterized, easy to manipulate and grow well in cell culture)
5. Induced stable expression in neuronal cells
6. Viral mediated expression system for mammalian cells. (i.e. Adenovirus, vaccinia virus etc.)
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As you see, numerous factors affect the choice of the most suitable expression system. Different scientific goals in combination with the specific features of the protein of choice should be considered.
 

In order to continue this tutorial, choose one of the following options:
Overexpress BHR1 in MDCK cells using an inducible expression system.

Overexpress BHR1 using a baculovirus expression system.

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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