Team:ZJU-China/Project/TheGhostKit/GhostSponge

The Ghost Kit: Ghost Sponge

Background

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.

Experimental design

Non-denaturing trait

• Sponge assembly: Mix 20ul bacterial ghost, 10ul biotinylated aptamer, 20ul thrombin and 50ul 2* PBS buffer, 37℃ for 30min. Pull down by 8000 rpm, 1 min. Wash 3 times with 200ul PBS. During the last wash, save 20ul supernatant as Control. Re-suspend the sample with 480ul PBS buffer after the last wash;

• Expel thrombin into solution: Prepare 3 EP tubes and add 160ul Sponge (from the first step) to each. Add 10ul sense DNA aptamer, antisense DNA aptamer and DNase respectively, 37℃ for 30min;

• Test thrombin activity: Co-incubate 20 ul previous sample with 20ul GST-tagged protein containing a cleavage site, add 40ul cleavage buffer and incubate at 37℃ for 60min. Denature the sample. Run the gel and do Coomassie Blue Staining.

Recyclable trait

• After pull down, wash 3 times with PBS;

• Expel thrombin into solution using sense DNA aptamer;

• Wash 3 times with PBS;

• Heat at 50℃ for 2 min to disassociate remaining aptamer-thrombin complex;

• Centrifuge to harvest the resultant products;

• Do “Non-denaturing trait” section experiment.

Results

Lane 1 shows a ladder denoting protein mass weight; lane 2 is loaded with control sample; lane 3-5 are respectively loaded with samples treated by method 1), 2) and 3). Thrombin helps cleave our loaded protein into a 5 kD and its 27 kD GST tag.

 Among all 3 methods, 3) is the most efficient one. The putative problem of DNase contamination is proved not a key problem if the purity is not stringently demanded, as the amount of DNase we used is negligibly small.

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.