Question

2. It has been established that the so-called ‘ ZZ domain’, a synthetic IgG binding protein derived from tandem repeats

of the B domain of protein A, interacts with the Fc portion of IgG molecules. HOW would you confirm that this ‘ ZZ domain’ indeed binds to the Fc region of IgG and not to that of IgM or IgE

a.What would you establish as criteria for physiologically relevant binding of this ZZ domain to IgG Fc portion?

b.What would you use as ‘ligand’ to determine binding to the ‘ receptor’ , in this case IgG

c.How would you assure the specificity of the ZZ domain for the Fc portion of IgG and not that of other immunoglobulins?

3. The signaling cascade of the FAS receptor, a first step in the extrinsic apoptotic pathway, includes a variety of critical protein-protein interactions

a.How would you isolate the protein that interacts with the DD, or Death Domain, of the FAS R?

b.How would you modify the cDNA encoding this DD to identify other interacting proteins in subpopulations of lymphocytes that interacted with the FAS R in Fas ligand-stimulated cells, but NOT in resting, or unstimulated, lymphocyte cells.

c.How would you test the SPECIFICITY of the Fas R DD domain interactions with your identified interacting proteins to be sure they uniquely interact with the DD domain amino acid sequence?

Answer 1

3. The signaling cascade of the FAS receptor, a first step in the extrinsic apoptotic pathway, includes a variety of critical protein-protein interactions

a.How would you isolate the protein that interacts with the DD, or Death Domain, of the FAS R?

Ans.Proteins that interacts with the Death Domain, of the FAS R can be isolated by the following methods such as coimmunoprecipitation, cross-linking, molecular biological methods like protein probing and the two-hybrid system.

b.How would you modify the cDNA encoding this DD to identify other interacting proteins in subpopulations of lymphocytes that interacted with the FAS R in Fas ligand-stimulated cells, but NOT in resting, or unstimulated, lymphocyte cells.

Ans.Lymphocytes markers can be identified and after finding the sequence, design the method to place the desired vector in the plasmid having mutation which modify the desired gene by recombination, synthesize oligomer and formation of protein which bind to the receptor and than recognize.

c.How would you test the SPECIFICITY of the Fas R DD domain interactions with your identified interacting proteins to be sure they uniquely interact with the DD domain amino acid sequence?

Ans.

Steps for the specificity of the Fas R DD domain interactions with the identified interacting proteins are as follows;

1.Production of a recombinant GST-hFasR fusion protein

2.Bacteria and Phages

3.Phage display library screening

4.Preparation of phagemid DNA, sequencing, and identification of interacting Death Domain

Verification of selected Death Domain-Fas R interactions