Guided example 2: does sheep ZAP70 have an active tyrosine kinase?


Melissa is working on a drug discovery programme to create new inhibitors of ZAP70 for use in autoimmune disease. Greg, a medicinal chemist in her group, has synthesised a series of protein tyrosine kinase inhibitors. She needs to devise a cellular assay to see which of Greg’s inhibitors inhibit the ZAP70 tyrosine kinase. She has ready access to primary T cells from the following animals:

  • Mouse
  • Rat
  • Sheep

To decide which of her models has a ZAP70 protein most closely related to the human one, she has decided to do a quick multiple sequence alignment. Using EBI search, she located the canonical sequences (isoform 1) for the human, mouse, rat and sheep ZAP70 proteins. For each one, she downloaded the protein sequence in FASTA format. She then performed a multiple sequence alignment of these proteins using the Clustal Omega tool. You can repeat her experiment if you like.

On the basis of this alignment, Melissa has decided to use sheep T cells for her assay but she wants to double check that sheep ZAP70 has an active tyrosine kinase domain before wasting time on developing an assay. For this we’re going to use a tool called InterProScan. This tool compares your chosen sequence with all the sequences in InterPro, a resource that provides functional analysis of protein sequences by classifying them into families and predicting the presence of domains and important sites. If you would like to learn more about InterPro before continuing, you can read the InterPro Quick Tour or follow the InterPro tutorial. If you are short on time you can just read the section on sequence searching in the InterPro tutorial.

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