Project Design

Overview:

Our aim is to prove that therapeutic protein biologic production in the edible, yet acido-thermophilic microalgae Cyanidioschyzon merolae will provide a purification independent, oral alternative to the injection dependent therapeutics currently produced. Protein therapeutics include: mono and polyclonal antibodies, erythropoietin, interferons, DNases, protein antibiotics, and in the case of Algulin: insulin (Leung, 2007). The largest hurdle of oral therapeutics administration is the passage through the stomach acid as it degrades a substantial amount of therapeutic administered. By taking advantage of C. merolae’s innate acidophilic membrane, such proteins should remain native within the microalgae, where the carbohydrate based cell wall of C. merolae will be degraded in the small intestine by endogenous enzymes and bacteria, releasing the therapeutic protein for absorption by the absorptive cells through to the portal vein.

In developing Algulin and considering patient-doctor relationships concerning diabetes, we decided to pursue a photobioreactor, optimized for production of various insulin types in C. merolae. We are developing minimal viable prototypes for the photobioreactor and are continuing to produce our insulin constructs and photo-optimization parts. As we started our project hoping to use Chlamydomonas reinhardtii as a chassis, our constructs are currently codon optimized for this microalgae as opposed to our final, viable chassis, C. merolae.

See here for our Composite Parts page here. For additional information see our Parts Overview page. In total, we are using seven registry parts, four new parts, and have improved two new parts for our development of Algulin.

Design principles for our project

Provide an acid resistant chassis for the administration of a protein therapeutic.
Design of a photobioreactor, achieved through rapid prototyping.
A modular insulin that can be characterized by fluorescence for safe compounding, tested by a binding assay with the insulin receptor.
A commercial potential product, for democratized distribution.

Our four new parts are as follows:

1. Insulin Protein Receptor (BBa_K3237010)

This human protein was put under constitutive expression for use in our in vitro studies. The receptor can be found in pancreatic cells and regulates blood glucose levels by its interaction with insulin proteins (Lee and Pilch, 1994) For purification purposes a N-terminal hexahistidine tag was added to the construct. Using this protein allows for us to study our protein in their active form. As we have designed our constructs to be able to remove the Red Fluorescent Protein and Cell Penetrating Peptide with furin protease, as well as allowing for full processing of the protein into a bioactive form using the same protease we hope to confirm that the insulin is bioactive after our experimental process. The purified receptor protein is mixed with purified and processed insulin and then run under a microscale thermophoresis assay (MST). This assay detects binding events between the insulin receptor and the insulin proteins, confirming that can theoretically be used for therapeutic use.

2. His-mRFP1-SCI57 (BBa_K3237019)

SCI57 is one of our chosen insulin variants. SCI57 is an ultrastable single chain insulin that therefore requires no secondary processing after translation like proinsulin (Hua et al., 2008). As with our other designed constructs, SCI57 has been fused to the mRFP1 protein which in turn has a hexahistidine tag on its N-terminus. For this construct we have also taken advantage of the furin protease and have added a furin cut site (with a GC linker) between the mRFP1-SCI57 fusion to allow for mRFP1 removal, leaving SCI57 in a bioactive form that will not be hindered by the presence of mRFP1. The addition of this protein to the registry will give not only variety of insulin constructs to the registry but can also be used to compare other single chain insulin designs.

3. NAB1 (BBa_K3237024)

Our main premise for the use of NAB1 is to analyze the change in algal biomass when manipulating the light antenna. Efficient growth of an algal population is important when proposing it as a therapeutic method, as the doubling time of microalgae is drastically slower as compared to E. coli (~8hours compared to ~20minutes respectively). NAB1 is an RNA binding protein that decreases translation of antenna proteins (Mussgnug et al., 2005; Beckman et al., 2009). Higher NAB1 expression causes smaller antenna size which therefore allows for the algae to tolerate higher light. As we hope to decrease light energy needed for growth this construct is used as a comparison to our antisense construct as described below.
This part in particular was codon optimized for use in C. reinhardtii and was not modified with synthetic tags or fusions.

4. NAB1 antisense (BBA_K3237012)

As described above the NAB1 antisense construct acts upon this RNA binding protein. This short DNA sequence is homologous to the NAB1 gene and is used to silence NAB1 expression. The NAB1 antisense DNA is transcribed into RNA. RNA interference (RNAi) is then used to prevent translation where the mRNA of the NAB1 gene is bound to the antisense RNA construct and initiates activity of the RISC complex in the host (Grishok, 2005). The RNA is then cleaved and the NAB1 translation is drastically decreased. This decrease in NAB1 causes the antenna to remain larger in size. With the longer antenna, less photons are needed for energy and less light is therefore needed for sufficient growth.

5. Constructs for the implementation of a kill switch

The use of microalgae as a chassis can promote a change in how we think about producing protein therapeutics. However, being that microalgae have a broad tolerance to environmental conditions there is a risk in the chance of environmental release. Release of insulin producing microalgae could be a large health and environmental risk so we sought to mediate any potential issues in the future when our system may be implemented. For this reason we modified a kill switch first proposed by Čelešnik et al., 2016. A kill switch using a nonspecific DNA/RNA nuclease nucA (BBA_K3237029) and a specific nucA nuclease inhibitor, nuiA9BBa_K3237028) was created.

The nuclease inhibitor nuiA is placed under a metal ion promoter PcopM72 (BBa_K3237027) which is a variant of the CopMRS operon (Čelešnik etal., 2016). Expression is kept on by the presence of copper, zinc or other metal ions at a supplemented concentration. This is able to continually inhibit the nucA gene which is under a constitutive promoter. Once the metal ion presence is insufficient, the inhibitor is turned off and the nucA that is being continually expressed will accumulate in the cell and cause cell death. As nuiA is under an inducible promoter, leaky expression will not affect the cells as it promotes living conditions; this may slow down the expression of nucA when cell death must occur, but due to the constitutive expression the cell will still accumulate nuclease protein and die due to the continual DNA/RNA cleavage.

Our improved parts are as follows:

1. Highly engineered mutant of red fluorescent protein from Discosoma striata (coral) (BBa_E1010)

Our use of red fluorescent protein is to provide standardization of insulin being produced in microalgae. As we wanted to eliminate the need for insulin extraction we needed a way to determine the quantity of insulin being produced over time and at the point of oral administration. For this reason we worked with an improved BBa_E1010. Our main improvement was the addition of a his tag. Although we propose no extraction methods we still wanted to conduct in vitro studies on our proposed synthetic, insulin constructs in E. coli. And with that looking at just the mRFP1 during activity assays and purification was determined to be a good control for our work.

With the studies in E. coli and ensuring comparative results, our constructs were all built with this His-mRFP1 fusion, whether we proposed extraction or not. The addition of the his tag allowed us to purify the proteins effectively in which afterwards the His-mRFP1 was removed from the insulin after cutting with the Furin protease. In summary this improved allowed us to:

Purify constructs from E. coli for activity assays and;
Provide a viable control for assay work in vitro and expression standardization in vivo for both E. coli and our microalgae chassis.

2. Proinsulin (modified protease recognition sites)

The main construction of this part was retained as the furin cut sites that replaced the original protease sites still allowed for proper protein processing. To be used in our project specifically we added the His-mRFP1 fusion to the N-terminus of the protein and added another furin cut site and the linker between the mRFP1 and proinsulin for removal during processing into the bioactive form. Our construct designs include different proinsulin constructs which therefore required of an additional improvement were a cell penetrating peptide was added to the C-terminus of proinsulin (which can be removed by furin as well). This peptide known as a protein transduction domain has been known to provide uptake into the intestinal wall and therefore entrance into the bloodstream (Liang and Yang 2005).

With the intent of characterizing our insulin parts, we designed them for both microalgae and Escherichia coli. Given microalgae grows much slower, E. coli provides an opportunity to explore which insulin constructs act as we expected. Additionally, it allowed us to produce an insulin receptor construct so that we could design an insulin-insulin receptor based activity assay using microscale thermophoresis. All purification of E. coli constructs were performed on an Akta Pure system.

Future Directions:

Complete characterization of parts
Complete expression of parts in C. merolae and refine transformation protocols
Complete NAB1 gene regulation system
Complete photobioreactor and fluorometer

References:

Beckmann, J., lehr, F., Finazzi, G., Kankamer, B., Posten, C., Wobbe, L., and Kruse, O. (2009) Improvement of light to biomass conversion by de-regulation of light-harvesting protein translation in Chlamydomonas reinhardtii. Journal of Biotechnology. 142, 70-77

Čelešnik, H., Tanšek, A., Tahirović, A., Vižintin, A,. Mustar, J., Vidmar, V., and Dolinar, M. (2016) Biosafety of biotechnologically important microalgae: intrinsicsuicide switch implementation in cyanobacteriumSynechocystissp.PCC 6803. The Company of Biologists. 5, 519-528.

Grishok, A. (2005) RNAi mechanisms in Caenorhabditis elegans. FEBS letters. 579, https://doi.org/10.1016/j.febslet.2005.08.001

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

Lee, J., and Pilch, p. (1994) The insulin receptor: structure, function and signaling. American Journal of Physiology. 266:319-334.

Leung W.W.F. 2007. Centrifugal Separations in Biotechnology

Liang, J., and Yang, V. (2005) Insulin-cell penetrating peptide hybrids with improved intestinal absorption efficiency. Communications. 335, 734-738.

Mussgnug, J., Wobbe, L., Elles, I., Claus, C., Hamilton, M., Fink, A., Kahmann, U., Kapazoglou, A., Mullineaux, C., Hippler, M., Nickelsen, J., Nixon, P., and Kruse, O. (2005) NAB1 Is an RNA Binding Protein Involved in theLight-Regulated Differential Expression of theLight-Harvesting Antenna of Chlamydomonas reinhardtii. The Plant Cell. 17, 3409-3421

Team:Lethbridge/Design