Bioinformatics

See instructions in attachment. Please note that each question is 3 pages excluding references Question 1 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: Number Protein Accession Number 1 E2F1 Q01094 2 E2F2 Q14209 3 E2F3 O00716 4 E2F4 Q16254 5 E2F5 Q15329 6 E2F6 O75461 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 cloning will 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. The following data is required in the report: 1. Multiple Sequence Alignment of all 6 proteins You can do this using Clustal Omega http://www.ebi.ac.uk/Tools/msa/clustalo/ 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 and state 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 the E2F4 protein (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 smaller stretch 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 molecule such as keyhole 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. Question 2: THE SWEATY PALMS MISHAP You have just begun a new job in a toxins laboratory developing antidotes against a range of protein toxins from various sources. On your first day, you are given a rack of 3 tubes containing toxins of one sort or another. You are asked to determine some of the chemical and functional properties of the molecules so antidotes may be designed accordingly. As you look curiously at the contents of each tube, you suddenly realise your sweaty palms have rubbed off the labels! Luckily for you, the boss is away for 3 days, which gives you enough time to determine the identity of the proteins by using a combination of protein identification techniques and bioinformatics. To begin, you subject the samples to Edman degradation and acquire the following sequences. Protein1: LysAspThrLysLeuGlyAsnTyrAspAsnValArgValGluPheLysAsnLysAspLeuAlaAsp LysTyrLysAspLysTyrValAsp Protein2: ValIleThrLeuGluAsnSerTrpGlyArgLeuSerThrAlaIleGlnGluSerAsnGlnGlyAlaPheAlaSerProIleGlnLeuGlnArgArgAsnGlySerLysPhe Protein3: AspTyrThrAspTyrGlyCysTyrCysGlyLysGlyGlyArgGlyThrProValAspAspLeuAsp ArgCysCysGlnValHisAspGluCysTyrGlyGluAlaGluLysArgLeuGlyCysSerProPhe ValThrLeuTyrSerTrpLysCysTyrGlyLysAlaProSerCysAsn You can now use this sequence information to determine the likely identity of the protein. Using the bioinformatics tools that you have learnt so far, you proceed to find the relevant biological information for each protein. Using this information, prepare a one- page information sheet for each protein. The following information must be in your report: 1. Protein Identity Provide your best guess for the identities of the proteins you had in each tube, including the biological source. What bioinformatics tool did you use for this purpose? How did you determine the identity of your protein? What factors did you consider? For each protein, indicate how confident you are of your identification. 2. Functional Properties What are the predicted subcellular localization, molecular weight and significant domains (if any) of each protein? Can you then find articles or textbooks to verify or contradict these predictions? Look for information on domain maps, function and size. Even if no known information exists for any of the proteins, state this. 3. Structural Characteristics What is the structure of each protein (if available)? If not, has a crystal structure of a similar molecule been discovered? What method was used to determine the structure? Include the structures, where possible, in your report in a neat, presentable and labeled format. 4. Antidote design Based on the information you have acquired on each protein, suggest a mechanism by which the protein action can be inhibited.