Amyloid-Beta and Tau are proteins which are known to be linked to the development of Alzheimer's disease, though the cause of the disease is still unknown [1]. They are both found in the brain in and around our neurons, and during Alzheimer's disease they accumulate to form plaques and tangles [2, 3]. The study of these aggregates are often done by staining them(4), which in turn demands an amount of the plaques great enough for staining. To instead study the early development of the aggregates a new method has to be developed. This could be studied by fusing a fluorescent markers like Green Fluorescent Protein (GFP) with the aggregation prone proteins. The problem is that when these marker are combined with the aggregation prone protein they become hard to express in Escherichia coli [5]. Therefore this year’s project is to optimize the expression the fusion proteins containing a fluorescent marker and amyloid-beta or tau in E. Coli.

To optimize the expression of the proteins we will be using overexpressed chaperones. Chaperones are proteins that can help other proteins to fold into their native three-dimensional shape, for example by dissolving aggregates and guiding both unfolded and misfolded proteins to their correctly folded form [6]. During our project we will be using four different chaperones and we will see how different combinations of these affect the expression of Amyloid-beta and Tau respectively.

Quick facts about Alzheimer's disease:

• Around 60-70 % of people with dementia have Alzheimer’s disease.
• Around 1 in 100 has Alzheimer’s disease in Sweden.
• The cause of Alzheimer’s disease is formation of protein accumulations in the brain.
• Alzheimer’s disease is a fatal disease.
• Read more here

The chaperones we will study are GroEL, GroES, DnaK and Trigger factor. GroEL and GroES helps unfolded and misfolded proteins to fold correctly [7]. DnaK helps with disaggregating and with unfolding of misfolded proteins [6]. And Trigger factor helps the protein to remain unfolded during its synthesis [7].

To overexpress the chaperones in the bacteria we will use a large plasmid containing all four chaperones and give them unique positively regulated promotors, one for GroEL-GroES, one for DnaK and one for TF. The purpose of this was to easily be able to induce transcription of the specific chaperones in different combinations.

The fluorescent markers we will fuse our proteins with are Enhanced-GFP (EGFP) and mNeonGreen. EGFP are a variation of GFP and mNeonGreen is derived from LanYFP [8]. mNeonGreen itself foldes much faster than EGFP and therefore one of these two could be expressed more easily than the other.

For every one of alzheimer-fluorescent fusion protein we will create a plasmid resulting in 4 combinations. These 4 plasmids will use a fourth promotor so that we can control this expression as well.

If we succeeds with our project we will create a model for successfully expressing these aggregate prone fusion proteins. The future of the project would then be to apply this model to other troublesome proteins and get a deeper knowledge about how the chaperones handle them.

Our work process

The overview of the project is summarized in figure 1 below. As can be seen in the figure, chaperones and conditions will be tested in a screening process, with the purpose to find the best setup for further detailed studies. The detailed studies will involve modeling with systems biology, where the modeling team will give suggestions on what the laboratory team shall do to achieve the desired task. The flow of data between the laboratory and the modeling team will eventually result in the final data, with is the optimized setup for the expression of our peptides.