A lot of the interest in apoptosis mediation has focused on serine proteases, but research is at a very early stage so little is known about how these enzymes affect apoptosis
One of the most studied areas in life science research today is apoptosis.
More specifically, as a mechanism playing an essential role in survival of an organism, apoptosis is providing an opportunity for novel therapeutics, notably for anti-cancer drugs.
The key issue is identifying targets and then understanding what effect they have in controlling apoptosis.
It is in this aspect that Promega's ProteoLink in vitro expression cloning system has been assisting one group at Cancer Research UK.
A lot of the interest in apoptosis mediation has focused on serine proteases, but research is at a very early stage so little is known about how these enzymes affect apoptosis.
Miguel Martins's group at Cancer Research UK is looking at one particular protease called Omi/HtrA2.
So far results have shown that this enzyme can process itself, but no other substrates have been identified.
If other substrates can be identified, Omi/HtrA2 will be implicated in a key stage in apoptosis.
In order to identify a substrate (or indeed several), the entire cell proteome has to be screened.
More importantly, there needs to be a facility to link proteomic information bank to gene sequence data; but traditional methods (such as 2D gel analysis or yeast two-hydrid screening) fail in this critical aspect.
In vitro translation, however, addresses this problem. Promega's ProteoLink system provides a fast convenient method that avoids the time-consuming steps of protein purification, peptide sequencing and cDNA isolation.
The adaptability of the method to a robotic platform enabled Miguel Martins's group to rapidly and consistently process several rounds of 96-well mini preps.
From the initial screen, eight positive pools were detected which were further refined to a single pool upon re-analysis with a lower protein loading (to avoid non-specific digestion).
This pool was then analysed to yield a single positive protein among the cDNA clones.
Work is currently on-going to characterise this positive protein to see if it fulfils the expected criteria for a Omi/HtrA2 target which, in turn, will provide insight into the role this protease plays in apoptosis.
