Case Study 1 - THE CURIOUS CLONING CONUNDRUM

Your mission is to make one antibody that recognises all the E2F proteins, E2F1, E2F2, E2F3, E2F4, E2F5 and E2F6. You are provided with a frozen sample of cDNA of E2F4 to work with in the laboratory. Before you do so, however, you need to work out which region of this protein will be used as an antigen.

The accession numbers for these proteins are as follows:

The first step in making an antibody is to make a recombinant antigen. This means that you should choose a region of E2F4 DNA that can be cloned into a bacterial vector and then expressd in E. coli. The recombinant E2F4 can then be purified by a variety of methods and injected into a mouse for antibody production. (Hint: Looking up antibody production methods and cloningwill help you understand the requirements of this case study).

Use all the bioinformatics tools that you know of to identify the region of E2F4 that will be used for the antibody production. You will then generate a report (No more than 3-pages, excluding references).

The following data is required in your report:

1. Multiple Sequence Alignment of all 6 proteins
You can do this using Clustal Omega http://www.ebi.ac.uk/Tools/msa/clustalo/- this is probably new to you, but works like BLAST, in that it aligns sequences. The difference is that it can align more than 2 sequences, all at once.

Your selection criterion is NO MORE THAN 3 RESIDUES IN A ROW (≥ 3) without identity (*) or high similarity (:).

Present a full, annotated multiple sequence alignment showing the region of interest.What is the importance of this step?

2. Epitope mapping - finding an antigenic peptide
As you now have the region you could potentially produce recombinantly, check if it is predicted to be antigenic. There are several tools available for antigenicity prediction. You may use whichever you find most appropriate.Comment on your choice.

3. 3D representation of region of interest
VMD can be used to visualise the region to be cloned. Find a crystal structure of theE2F4protein (can be whole or partial, standalone or bound to another protein) and using VMD, highlight the region that you have indicated above. What does your 3D representation tell you about the region you have chosen? Can you think of two functions for this part of the protein?
There may be a smaller stretch of amino acids in this region of E2F4 that was extremely homologous to the other E2F family members, if so, highlight this in a different colour. Can you think of a reason why this smallerstretch is so homologous?

4. Functional information 
Present a functional map of E2F4 (either published or self-made). Look up some published articles with references to E2F4 and use appropriate bioinformatics tools for information about functional domains. Are your predictions about a possible function for both the region and the smaller stretch correct?

5. Complexing 
When injecting small antigens into an animal to elicit an immune response, it is common to conjugate the antigen to a larger protein. A common method is to covalently attach them to a larger moleculesuch askeyhole limpet hemocyanin (KLH). Determine the molecular weight of the region you have chosen, and make a comment about whether you should conjugate your antigen and why this is necessary.

6. Region to Clone
All of these tests and tools have been looking at the protein sequence of E2F4. However,in order to produce a recombinant protein, you have to create a bacterial DNA vector with the E2F4 DNA for your region of interest cloned in. Furthermore, you only want to clone in the sequence of DNA that codes for the region of E2F4 you have selected.Show the E2F4 gene sequence, highlighting the part of the sequence that codes for the region you wish to produce.

Other important information:

• This report is to be done independently and is worth 15% of your mark for the BIO5INF unit. You are free to format your report as you see fit. Going over the specified limit will incur deductions. Any evidence of plagiarism will be dealt with harshly.

• The report is due on Friday 6th October, no later than 5pm. A clearly labelled, Turnitin submission inbox that will be made available on LMS. Late submission penalty of 5% per day after applies after the deadline. A hard-copy submission is not required.

• Ensure that your report is succinct but covers all important points to understand the problem and the solution you have uncovered.

• Remember to include brief figure legends and provide referenceswhere necessary.