Diabetes is a metabolic disease that results in hyperglycemia due to a defect in insulin secretion, insulin action or both. In the former, the β cells of the pancreas are destroyed by the immune system and no insulin is secreted which results in type 1 diabetes. In the latter, the β cells secrete insulin but the individual develops an insulin ‘resistance’ which results in type 2 diabetes. We developed PRISMO, an alternative therapeutic system that can treat both cases.[1][1]

What is PRISMO?

PRISMO, short for probiotic insulin secreting modified organism is a bacterium that was designed to colonize in the gut of diabetic patients and secrete insulin like peptides when induced. In type 1 diabetics it will do what the β cells in the pancreas can’t do and in type 2 diabetics it will be another insulin source in addition to the β cells.

How it works?

We aim for PRISMO to be an inducible system that will secrete insulin to treat diabetic patients. PRISMO will be induced with arabinose, a 5 carbon sugar that is also used as a food additive. Arabinose is also not absorbed by the intestine which assures that the sugar will reach the gut, it is also a potential prebiotic. The patient will ingest some amount of arabinose after meals and PRISMO will secrete insulin from the gut.

Single Chain Insulin Analogs

Wild-type human insulin consists of 2 chains, the A chain and the B chain. This hormone is a product of a single chain precursor called proinsulin which has an additional C chain that connects the A and B chains. This C chain is cleaved after the A and B chains are connected via disulfide bonds [2].

Since wild-type E. coli can’t make the C peptide cleavage, instead of wild-type insulin PRISMO secretes single chain insulin analogs, abbreviated as SCIs. These SCI analogs have short linker sequences instead of the C peptide which promotes the folding of the peptide. Wild-type insulin and SCI analogs have similar binding affinities to the insulin receptor.

To find the best SCI candidate we searched the literature and stumbled upon an article called ‘Single-Chain Insulins as Receptor Agonists’. Here the researchers used directed mutagenesis to generate distinct SCI constructs and found that some linkers contributed to SCI bioactivity. They measured the activities of 36 different linker sequences including the full C-peptide and no linker at all. Out of all the linkers we chose the most active 2 to add to our constructs [3]. We also added another linker from a patent published in 1996 about single chain insulin analogs [4]. We also came across an article published in 2008 which modified the A and B chains of insulin in single chain insulin molecules to increase the bioactivity. They changed several amino acids in the sequence of A and B chains to decrease the pI (isoelectronic point) of the SCI, making it closer to the one of wild type insulin. They also created a linker sequence by predicting effects of amino acid changes [5]. Thus, in total we have 4 different linkers and 2 variations of the A and B chain. We paired each linker with the modified A and B chain and with the wild-type producing 8 different SCI constructs.

Ag43 TEV Release System

The PRISMO release system works by utilizing the Ag43 autotransporter and the TEV protease. The translated SCI peptides are directed towards the periplasmic membrane via the pelB signal sequence. The SCI peptides are fused with a TEV protease cut site and are displayed on the surface of the cell via Ag43 autotransporters continuously (with a constitutive promoter). Along with the SCI analogs TEV proteases are also displayed on the surface of the cell when induced with arabinose, again via the Ag43 autotransporter. Due to the fluid mosaic model of the cell membrane these autotransporters move and collide with each other in the cell membrane. If these collisions are in the right position and angle the TEV proteases cleave the SCI analogs from the autoransporter at the fused TEV protease site releasing them into the medium. To test the release system we created a construct which has sfGFP instead of the SCI. We were then able to quantify the protein release via a M5 spectrophotometer [6]. [6].

Cell Penetrating Peptides

PRISMO colonizes in the gut and also secretes the SCI analogs into the gut but for these SCIs to be effective they have to get into the bloodstream. To achieve this we fused a cell penetrating peptide to our SCIs called PenetraMax. This peptide allows our SCI peptide to penetrate the intestinal epithelium to enter the bloodstream [7].

Cell Strains

In our project we integrated 4 different E. coli strains into our project. First was the DH5-Alpha E. coli strain, we used this strain for our cloning experiments. It was a good candidate for our cloning experiments because of its high transformation efficiency [8]. They have 3 defined mutations which help with plasmid insertion and one that allows blue-white screening [8]. We used the BL21 for protein expression, due characteristics such as protease deficiency, low acetate production at a high level of glucose, and enhanced permeability (probably due to a simple cell surface) make E. coli BL21 a desirable host for the production of genetically engineered proteins [9]. The BL21 Marionette strain is a modified version of BL21, we used it for the expression of protein which were induced via arabinose. The BL21 Marionette strain has 12 different optimized small sensors integrated into the genome [10]. Finally the chassis of our project is the Nisille strain of E. coli, we use Nisilee because it is common probiotic organism [11].

References

1.Diagnosis and Classification of Diabetes Mellitus American Diabetes Association Diabetes Care Jan 2013, 36 (Supplement 1) S67-S74; DOI: 10.2337/dc13-S067

2.Fu, Z., Gilbert, E. R., & Liu, D. (2013). Regulation of insulin synthesis and secretion and pancreatic Beta-cell dysfunction in diabetes. Current Diabetes Reviews, 9(1), 25–53. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934755/

3.Rajpal, G., Liu, M., Zhang, Y., & Arvan, P. (2009). Single-chain insulins as receptor agonists. Molecular endocrinology (Baltimore, Md.), 23(5), 679–688. doi:10.1210/me.2008-0349

4.Chance, R. E., Dimarchi, R. D., Hoffmann, J. A., Long, H. B., & Miller, A. R. (n.d.).

5.Hua, Q. X., Nakagawa, S. H., Jia, W., Huang, K., Phillips, N. B., Hu, S. Q., & Weiss, M. A. (2008). Design of an active ultrastable single-chain insulin analog: synthesis, structure, and therapeutic implications. The Journal of biological chemistry, 283(21), 14703–14716. doi:10.1074/jbc.M800313200.

ACS Synth. Biol.201984686-696 Publication Date:February 27, 2019 https://doi.org/10.1021/acssynbio.9b00062

6.Khafagy, E.-S., Kamei, N., Nielsen, E. J. B., Nishio, R., & Takeda-Morishita, M. (2013). One-month subchronic toxicity study of cell-penetrating peptides for insulin nasal delivery in rats. European Journal of Pharmaceutics and Biopharmaceutics, 85(3), 736–743. https://doi.org/10.1016/j.ejpb.2013.09.014

7.Chan, W. T., Verma, C. S., Lane, D. P., & Gan, S. K. (2013). A comparison and optimization of methods and factors affecting the transformation of Escherichia coli. Bioscience reports, 33(6), e00086. doi:10.1042/BSR20130098

8.Jeong, H., Barbe, V., Lee, C. H., Vallenet, D., Yu, D. S., Choi, S.-H., … Kim, J. F. (2009). Genome Sequences of Escherichia coli B strains REL606 and BL21(DE3). Journal of Molecular Biology, 394(4), 644–652. https://doi.org/10.1016/j.jmb.2009.09.052

9.Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2018). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 15(2), 196–204. https://doi.org/10.1038/s41589-018-0168-

10.Sonnenborn, U. (2016). Escherichia colistrain Nissle 1917—from bench to bedside and back: history of a specialEscherichia colistrain with probiotic properties. FEMS Microbiology Letters, 363(19), fnw212. https://doi.org/10.1093/femsle/fnw212