Since our whole project is based on the ability of Carboxysomes to retain its metabolic properties in vitro, an easy purification, one that you can standardize for industrial production and that can produce a high yield of your protein complex, is needed.

In order to achieve this, we thought about different approaches:

1) Sucrose Gradient centrifugation

From the literature, we knew that Carboxysomes can be purified using traditional methods such us a sucrose gradient centrifugation (1). In this protocol, the cells are disrupted by sonication; then they are centrifuged and resuspended with different buffers in several steps. The Carboxysome-enriched solution is loaded onto 10 to 50% (wt/vol) linear sucrose density gradients. After that, the prominent band near the middle can be isolated and should contain the Carboxysomes.

Yet, since the proteins get separated according to size and density, the specificity of this technique is not very high and is most likely submitted to human error.

2) Magnetic Carboxysome

During 2011, the Dundee iGEM team worked with Pdu, another bacterial microcompartment.

Among other things, they tried to make Pdu magnetic for bioremediation processes or easier recovery of modified microorganisms. To achieve this, they attached bacterioferritin (Bfr) from Escherichia coli to Pdu40, a tag for destination to the inside of Pdu. Bfr forms a large storage protein for iron and iron oxides. This should concentrate the particles inside the BMC and make it simple to separate with a magnet (2).

We thought about attaching a protein such as Bfr to one of the proteins of the Carboxysome’s shell in order for the microcompartment to be coated with metallic particles and thus, be able to be purified using its magnetic properties. However, proteins from magnetosome associated systems or from the electron transport chain (ferritin, etc.) are too big and complex to be used for such a purpose.

So, after searching for other mechanisms of high yield purification, we decided to mainly focus on different affinity chromatography.

3) Biotinylation

In 2010, the Freiburg Bioware iGEM team designed Virus Construction Kit for Therapy. In their project, they tried two different methodologies for the purification of viruses’ capsids. One of them was based on HisTag affinity purification and the other based on biotinylation of capsid.

According to their results the detection limit of the biotinylated viral capsids was about 10fold more sensitive (4). The principal advantages of this procedure is that with this mechanism you can isolate a protein complex in an assembled way and have the protein aggregate ready for further analysis; it also shows good yield results.

How does biotinylation works?

In nature, E. coli biotin holoenzyme synthetase, BirA, catalyzes the transfer of biotin to the epsilon amino group of a specific lysine residue of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase (4).

Schatz et al (5). Identified the 15 amino acid peptide 5’ GLNDIFEAQKIEWHE 3’ as the most efficient sequence to produce biotinylation. So, added to any protein of interest and in the presence of the BirA gene it can cause the protein to be selectively tagged.

Using this approach, we learnt from our host lab PSBL about the INTACT (isolation of nuclei tagged in specific cell types) method for the purification of nuclei from plants (6). This technique purifies biotin-labeled nuclei using streptavidin-coated magnetic beads, without the need for specialized equipment. In it, you generate a nuclear targeting fusion protein, which consists of three parts: a specific domain for nuclear envelope association in plants, a green fluorescent protein (GFP) for visualization and the biotin ligase recognition peptide. The expression of this construct and the BirA gene in plants allows purification of specific nuclei.

The INTACT method supposedly takes 2 days and should be adaptable to any organism that is amenable to transformation. So, we decided to combine both:

• - The protocol for the sucrose gradient centrifugation (early steps): Disruption and centrifugation steps and the use of the same buffers in which the Carboxysome is stable.
• - The INTACT protocol (biotinylation and later steps): Fluorescence visualization and streptavidin- coated magnetic beads isolation process.

With this, we were able to create a new methodology for the extraction and purification of Carboxysomes.

The general idea was to use the preview construct C.RFP (for more information about this construct, go to the Formation section) and add to the RFP the biotinylation acceptor peptide GLNDIFEAQKIEWHE. This way we could visualize the microcompartment using microscopy (to check for formation and correct induction) and allow Carboxysomes to get affinity bound to streptavidin beads.

The characterization of the purification system needs to be tested in more detail:

• - The improvement on the efficiency between using traditional methodologies and our innovative system of purification.
• - A quantification of the obtained yield of functional and assembled Carboxysomes.

We also need to verify if this system can be scalable for industrial production by checking costs and advantages of the production of the in vitro Carboxysomes.