Team:Lethbridge/Demonstrate

Demonstrate

The expression of insulin in microalgae has never been accomplished before. Although this is a great opportunity to start the discussion of different ways we can deploy and produce protein therapeutics, it has also meant that we have done a great deal of preliminary work for ensuring that our project is viable.

Chassis Determination

Our proposal for using microalgae as an oral alternative has met a great deal of challenges in picking the right microalgae chassis for our insulin. Starting out with our project, we first decided to work with the most prevalent microalgae species in both the food industry and in research. Our first hosts Spirulina (Arthrospira platensis) and Chlamydomonas reinhardtii have been in the market as a health food and have been a host system for research respectively. We decided to check if the microalgae would be able to withstand the harsh conditions of the stomach, as we needed it to be stable and undigested until it hits the lower intestine. However our results showed that these chassis we not ideal (for more information please refer to the Results page.)

Although this may be mediated by an enteric coated pill as used with other pharmaceuticals (Park et al., 1984), we wanted to increase our chances as much as possible to bypass the stomach. Furthermore, as we are at this point unsure of the expression levels and therefore algal mass needed for optimal dosage, we did not want to limit ourselves to the usage of these algal species. Luckily with a gift from the Rader lab at the University of Northern BC we were able to obtain a safe, acidophilic and potentially edible microalgae, Cyanidioschyzon merolae. Being that C. merolae is optimally grown at pH ~2.5, it was a perfect candidate for withstanding the stomach pH, which our results confirm.

Figure 1: C. merolae cultures placed in growth media modified to different pH points. The OD750 was measured over time. The blue line represents the population in media at pH 2.5 and the orange line represents the media at pH 7.0. n=3

The pH treatments of C. merolae confirmed that it would likely be stable in the acidic pH of the stomach, making it a perfect chassis for our work and proof of concept for our project.

Storage of Microalgae

The next step of our proposed project is determining in what way may we be able to store microalgae long term while ensuring that it is still able to withstand the harsh conditions of the stomach. We first moved to lyophilization but found that the cell wall and ultimately, the microalgae's stability in acidic conditions was compromised. Upon researching more into the technique it was determined that lyophilization, although routinely used in microalgae health food storage it is also used to primarily to break the cell walls for protein extraction (Ryckebosch et al., 2009). For more information please see our Results page.

Our second storage idea was to determine if storage in glycerol and then held at low temperatures would affect the microalgae. It should be noted that the intake of glycerol by diabetics does not affect glucose levels and is not harmful unless in incredibly large dosages (Thornit et al., 2009).

Figure 2: C.merolae cultures under different pH treatments over time. n=3

Our results showed that the initial amount of cells is less than that of the control that was not flash frozen. This may be due to the -80 storage as microalgae show a loss of integrity after long term low temperature storage (Aujero & Oseni, 1979). As to the overall impact of the flash freezing and then low pH Allen media, the graph shows that the populations at all pHs are stable over time. With statistical analysis using the student t-test, the Acidic treatment did cause a significant change in population over the four hour time period while the basic and neutral pH treatments did not cause significant change.

In summary, our preliminary studies show that the acidophilic C. merolae microalgae species is a better chassis than more highly studied species such as spirulina and C. reinhardtii. As well, we were able to determine that storage methods may include low temperature storage in sugar derivatives such as glycerol.

Insulin Activity

We were able to purify three proteins this season: mRFP-SCI57 (BBa_K3237018), mRFP1-Proinsulin (BBa_K3237016) and the insulin receptor protein (BBa_K2148013).

The first insulin derivative we chose as a proof of principle was the proinsulin with modified cut sites first proposed by Tsinhua 2014. With the insertion of the furin cut sites, our in vitro studies were easier as only one protease for insulin processing was needed and as it is a human protease (Hay and Dockerty, 2003) ensures that the proinsulin would be processed in vivo.

Our second insulin choice was SCI57, a super stable insulin analog that is also a single chain variant (Hua et al., 2008). Using the same strategy as the proinsulin part, we added a furin cut site to remove the RFP after purification. For information on our purification results please see our Results page.

Since the Furin protease was unable to cleave both the mRFP1-Proinsulin and mRFP1-SCI57 insulin proteins we decided to move ahead and test SCI57. As this insulin is a single chain there is a chance that it is still active despite the mRFP1 still being fused.

In order to determine if binding was occurring we ran both a MicroScale Thermophoresis assay (MST) and an electrophoretic mobility shift assay (EMSA) to confirm binding.

MST Analysis

MST analysis is a way to analyze biochemical interactions such as protein-nucleic acid and protein-protein interactions. This analysis tool is able to detect binding events at a nanomolar concentration that can be interpreted as interaction stoichiometry and dissociation constants (Plach et al., 2017). As such, we hoped to use this technology to detect if binding was occuring with our SCI57 insulin construct and the insulin receptor protein. Luckily, as RFP is still bound, we were able to use the fluorescence from the protein to detect our binding events.

Figure 3: A time scale graph showing the MicroScale Thermophoresis assay indicating protein-protein interactions between the mRFP1-SCI57 protein and the insulin receptor. The blue data indicates fluorescence reads with just the mRFP1-SCI57 construct at 500nM concentration. The green data represents the samples containing the mRFP1-SCI57 and insulin receptor at 500nM concentration and ~17,000nM receptor respectively. The increase in fluorescence reads with the mRFP1-SCi57 + insulin receptor protein indicates a binding event between the two proteins.

Our data shows that when mixed together there are binding events, and therefore a small shift, between the mRFP1 and Protein receptor. To further analyze this result we attempted to run an electrophoretic mobility shift assay, which was found to be inconclusive. More can be found on our Results page.

Overall, we have been able to express and purify two insulin proteins from E.coli and have attempted to test activity with the insulin activity with one protein. For algae, we have been able to find a suitable chassis for our work and have started optimizing storage conditions for microalgae for future work and for production storage that is safe for use and safe for diabetics.

Future directions

• Perform more Furin cleavage assays or determine what may be affecting the cutting efficiency.
• Although our data from the MST assay shows that there is binding events occurring we were unable to to do a test with a dilution series being that the fluorescence was halved during the binding. The receptor when bound to the insulin was able to shadow the fluorescent reads. In the future we hope to determine the binding events without RFP present and use a different fluorescent probe such a his tag binding dye.
• Finish creating all constructs and start expression experiments in C. merolae microalgae (after assessing molecular biology to ensure our current constructs are still feasible).
• Optimize storage testing and model the amount of population that would contribute to complete delivery of the insulin.
• Do more testing to determine if the microalgae are resistant to pepsins and other stomach/intestinal enzymes.

References

Aurojero, E., & Oseni, M. (1979). Viability of frozen algae used as food for larval penaeids. Aquaculture Department Quarterly Research Report, 3, 11-16

Hay, C., & Docherty, K. (2003). Enhanced expression of a furin-cleavable proinsulin. Journal of Molecular Endocrinology, 3, 597-607.

Hua, Q., Nakagawa, S., Jia, W., Huang, K., Philips, N., Hu, S., & Wiess, M. (2008). Design of an active ultrastable single-chain insulin analog: synthesis, structure, and therapeutic implications. The Journal of Biological Chemistry, 21, 1473-14716.

Park, H., Chernish, S., Rosenek, B., Brunelle, R., Hargrove, B., & Wellman, H. (1984). Gastic emptying of Enteric-Coated Tablets. Digestive Diseases and Sciences, 29, 207-212.

Plach, M., Grasser, K., & Schubert, T. (2017). MicroScale Thermophoresis as a tool to study Protein peptide interactions in the context of large eukaryotic protein complexes. Bio-Protocol. 10.21769/BioProtoc.2632

Ryckebosch, E., Muylaert, K,. & Foubert, I. (2011). Optimization of an analytical procedure for extraction of lipids from microalgae. Journal of the American Oil Chemists Society, 89, https://doi.org/10.1007/s11746-011-1903-z

Thornit, D., Sander, B., la Cour, M., & Lund-Anderson, H. (2009). The effects of peroral glycerol on plasma osmolarity in diabetic patients and healthy individuals. Basic & Clinical Pharmacology & Toxicity, 105, 289-293