mosquito Crystal for Protein Crystallisation Screening

Development of New Flu Drugs Using Protein Crystallography

Pandemic and world-wide emergence of Tamiflu-resistant seasonal human influenza has highlighted the need for the ongoing development of new anti-virals, efficient production of vaccine proteins and novel diagnostic tools. Analysis of drug-protein interactions using X-ray crystallography can be used to elucidate the key molecular contacts which lead to inhibition. These interactions can be optimised to establish strong anti-viral drugs. However this pioneering work requires large quantities of protein. Prof. Kurt Krause, Otago University, New Zealand and his group have been establishing a novel eukaryotic expression system for influenza neuraminidase (NA). Ashley Campbell, an MSc student has worked on expressing an NA protein in this system that is suitable for X-ray crystallographic studies.

Miniaturising the Crystallisation Process

The influenza surface glycoprotein NA is essential for the efficient spread of the virus. When inhibited, viral spread between hosts is limited and symptoms of the disease decrease rapidly. Miniaturising the optimal expression system for an NA protein has proved to be challenging. The initial stages required Ashley Campbell to set up small-scale crystal screens in order to find suitable conditions for crystallisation. Choosing these conditions can be a daunting experience when you need to conserve limited pure protein sample and set up very small and accurate drop sizes.

Using the mosquito® Crystal robot, crystallisation drop sizes were reduced to 150 nL, allowing more conditions to be screened. Ashley set up crystal drops using the hanging drop method on 96 well plates, containing various crystallisation conditions. The multi-aspirate function of mosquito Crystal allowed for the aspiration and mixing of 150 nL of protein sample with 150 nL of each crystallisation condition. Crystal hits occurred in 27 out of the 192 conditions (Fig 1a) allowing many opportunities to optimise the crystal (Fig 1b) from only a small protein sample.

Further X-ray diffraction of these crystals produced crystals into high 2Å ranges (Fig 2).

accuracy and precision Reducing dispensing volumes to nanolitre amounts requires accuracy and precision that is not possible using manual methods. Automating this process enables consistency in pipetting and minimise operator error. The volume range capabilities of liquid handling systems vary considerably and are dependent on on the type of dispensing technology. The accuracy of mosquito Crystal is unrivalled mainly due to its unique positive displacement pipetting. Prof. Krause commented, “The mosquito Crystal has made it possible to work with more proteins that are only available in small amounts. This means that new projects can be started more readily and new vista are opened up for scientific inquiry.”

It also boasts a system of disposable micropipette tips that reduce the likelihood of cross contamination, remove the need for time-consuming wash steps or replacing costly, fixed-tip dispenser heads. The pipette tip design also allows the tip to reach the bottom of a well – leaving minimal (less than 0.3 ?L) dead volume in the source well and no in-tip dead volume. This allows you to keep all the savings from an efficient set-up process.

From a student’s point of view Ashely Campbell commented, “I have found mosquito Crystal very user-friendly which has meant that I can work independently and still produce high quality crystals.”

Ashley Campbell is an MSc student in biochemistry, at the University of Otago. She works with Prof. Kurt Krause who is Director of Webster Centre for Infectious Diseases at the university. Ashley’s research focuses on the expression and purification of the influenza neuraminidase protein in a eukaryotic cell line for the identification and characterisation of novel inhibitory drugs.