Team:ZJU-China/Project/TheGhostKit/GhostSponge

The Ghost Kit: Ghost Sponge

Previously described methods concerning protein purification are of great diversity. However, few of them are designed as non-denaturing ones that only allow limited characterization of a distinct protein, as its activity is undermined and thus in need of an alternative method to detect.

Here we build a device on the basis of a fantastic combination of bacterial ghost and aptamer. The incredible chemistry between these two elements enables a high-efficacy non-denaturing purification and therefore greatly revolutionizes the protein purification methodology. It’s pretty handy, with the protein activity maximized. It’s definitely a COOL design. We call it‘Ghost sponge’.

Our device includes the following components – bacterial ghost, inner membrane scaffolds, biotinylated aptamers and our protein of interest – thrombin. Our previous experiments prove that:

• Scaffolds we build are powerful enough to be localized upon the inner membrane.
• Thrombin aptamer we use are of high efficiency.

Naturally we come to sound out the possibility of applying these amazing features into protein purification, if its high-affinity aptamers are available. Bacterial ghost has no cell content – a great characteristic for us as the disturbance in purification is partially erased. Nontheless, what we get is not solely a protein molecule, but a protein-aptamer complex. How we get rid of the unwanted aptamer becomes a trouble we are about to shoot.

Preliminarily, we come up with three approaches:

1. By adding the identical nucleotide sequence as aptamer to expel the protein into the solution through competitive binding;
2. By adding the complementary nucleotide sequence as aptamer to expel the protein into the solution through competitive binding;
3. By adding DNase to digest the aptamer.

Of all these three, the first method possesses another advantage – it's recyclable.If we heat the sample at 50℃ for 2 minutes to dissociate the protein-aptamer complex without significantly compromise the scaffolds we construct.

Although our method is aptamer-dependent, we totally believe that with the development of SELEX and other up-and-coming aptamer-screening methods, the novel purification method may serve as a powerful tool to study proteins with their activity remains.

Our device owns the following amazing features:

1. As aptamers are either DNA or RNA, they won’t interfere with protein of interest in gel analysis. Conversely, traditional antibodies may contribute to protein background and induce troubles in subsequent analysis.
2. The non-denaturing procedure significantly reserves protein activity by which the further study of endogenous proteins is enabled.
3. Although purification by virtue of aptamers is developed, its dependence on beads is a major problem in application. Our bead-free system release lab workers out of the dilemma of whether to buy beads.
4. The protocol is really handy and the product has a long shelf life.