Inhibiting the spread of influenza virus requires understanding the interactions between influenza proteins (e.g. surface glycoprotein neuraminidase (NA)) and drugs. These molecular contacts can be studied using X-ray crystallography as a powerful tool and be optimized to establish strong anti-viral drugs.
Technological advances in protein crystallization methods have made it possible to study the structure and interaction of protein and drugs in low volumes (requiring a smaller amount of sample) and with high-throughput. Automation of the liquid handling process for these high-throughput miniaturized assays (e.g. with the use of TTP Labtech’s mosquito® Crystal) can reduce operator error and provide better consistency.
One of the stories in our latest labCrystal journal describes the use of mosquito Crystal for screening various crystallization conditions for NA. The work was carried out by Ashley Campbell, an MSc student with Professor Kurt Krause, Otago University, New Zealand.
miniaturizing the crystallization process
Ashley has been working to establish a eukaryotic expression system for NA suitable for
X-ray crystallographic studies. Miniaturizing this system has proved to be challenging, but recent work using HEK cells has yielded successful results and sufficient protein for crystallization studies.
Using mosquito Crystal, crystallization drop sizes could be reduced to 150 nL, allowing more conditions to be screened. Ashley’s initial work produced crystal hits in 27 out of 192 conditions that were employed (Fig 1) allowing many opportunities to optimize the crystal from only a small protein sample. Further optimization produced crystals of an average of 200-250 µm which led to clean isotropic diffraction and well resolved spots into the high 2Å ranges.
Prof. Krause commented, “This system has made it possible to work with more easily attainable amounts of protein, which allows new projects to be started more readily, and can lead to new areas being opened up for scientific inquiry.”
Fig 1. Crystals of influenza neuroaminidase in a 300 nL drop, set up by mosquito Crystal in a crystal screen.
If you would like read the full article or find out about how other crystallographers have been using our liquid handlers please go to our resource section on our website www.ttplabtech.com.
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