Taster

The methods detailed below are aimed at secondary school and college students as a taster to the full Antisense Protocol. The method is based around antisense peptide screening to identify interactions between a target protein and other proteins. For this taster the target protein has the same name as a well known Simpson's character - Homer. This protein is found in the brain and has been suggested to play a role in stress, a very appropriate role for a compound named after Homer Simpson!

The method will use the
mRNA for Homer from the NCBI database to generate an antisense peptide that is then used to search within a protein database. Proteins with similarity to the antisense protein should bind to the homer protein and potentially have biological effects. In view of the protein chosen being named after Homer Simpson for this taster the protein databases to be searched will be from the fruit fly (Drosophila melanogaster), an organism that is bound to annoy Homer! This species is also a commonly used model in Biomedical research.

HomerFly


Method:

  • Step 1: Click on the link for the Taster Script, which will open in a new window, and copy the full text displayed. Then return to this window.

  • Step 2: Using the link for the Python Compiler, which will again open in a new window, paste the copied text from Step 1 into the compiler. Then return to this window.

  • Step 3: Click on the link to the Homer mRNA, which will open in a new window, and copy the full text displayed. Then return to this window.

  • Step 4: Return to the open Python compiler window, click the play button, type in the Homer followed by a return where it asks for a name and then paste in the text from Step 3 where it states mRNA. Copy the complete Antisense sequence generated, starting with a Y. Then return to this window.

  • Step 5: Click on the link to the BLAST program, which will open in a new window and paste the antisense sequence into the "Enter Query Sequence" box. Then return to this window.

  • Step 6: Adjusting the settings to perform the protein database search. Return to the open BLAST program window.

  • (a) In the "Job Title" box type Homer.

  • (b) In the "Choose Search Set" section make sure that "Standard databases (nr etc.): is selected.

  • (c) In the "Standard" section "Database" box select "Protein Data Bank proteins(pdb)" from the dropdown link.

  • (d) In the "Standard" section "Organism" box type "Drosophila melanogaster (taxid:7227)".

  • (e) In the "Standard" section "Exclude" section click the "Models (XM/XP)", "Non-redundant RefSeq proteins (WP)" and "Uncultured/environmental sample sequences" boxes.

BLAST1

  • Step 7: Adjusting the Algorithm settings. Return to the open BLAST program window.

  • (a) Click the "+ Algorithm parameters" title.

  • (b) In the "General Parameters" section "Expect threshold" enter "20,000"

  • (c) Click the "BLAST" button.

BLAST2
  • Step 8: When the results appear you will see the names of the Drosophila melanogaster proteins that Homer interacts with, the first of these has a name that is probably very representative of what Homer Simpson would want to do to a fruit fly! Using these results it is possible to create 3D models of the interaction.

TasterModel


Model of Homo sapiens Homer protein (red) bound to Drosophila melanogaster Dispatched protein (blue) generated using ClusPro.

A more detailed protocol for generating all the possible antisense peptides, searching protein databases, modelling interactions and interpreting the results is available at
Antisense Protocol.

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