Choosing proteins

Integrated Human Practice

On this page you can read about how we reached out to experts within the field of space medicine in order to learn more about the main health problems during space exploration.

Our findings has greatly impacted the purpose of our project, as it has:

• Helped us understand which problems we can solve and how
• Helped us conclude on a list of proteins that would be ideal for space related health issues
• Helped us to evaluate the limits of our protein production system with regard to target proteins

In parallel with developing our project we have been speaking with several experts within the field of health and space medicine. Given that protein therapeutics for astronauts isn't a very established field yet we have been very dependent on the input we have gotten from experts. The experience from the experts has been very important for choosing proteins for production with our system both from a medical and pharmaceutical point of view. The experts have been the very core of developing our project and making our product useful outside the laboratory which clearly show that we have integrated their thoughts and opinion into the purpose of our project.

Based on our research and discussion with experts we propose to use our system for production of IGF-1, PTH, GH, G-CSF and camelid antibody during long lasting space missions. First of all this decision is based on our interviews and discussions with experts. Based on their opinions we have further considered how easy the different proteins would be to produce with our system taking into consideration posttranslational modifications (PTM), protein size and the literature. We have therefore analyzed potential target proteins from several perspectives, especially considering 1) is this protein interesting from a medical point of view? 2) Is this proteins feasible to produce with our system? (for more information see the analysis below). It has been very important for the development of our project that we have been able to speak with experts in space medicine and protein production such as Virginia Wotring, Karen McDonald, Jon Scott, Jørgen Sauer, Michael Hecht and Eva Horn Møller (which we will introduce below) and we have very much incorporated their inputs into the very core of our project.

Experts. We chose to contact Virginia Wotring, Adjunct Associate Professor, Center for Space Medicine, Department of Pharmacology, Baylor College of Medicine, Houston, TX, US, because of her background as pharmacist and her experience with drugs and drug stability in outer space. Given that we wanted to produce protein drugs we wanted to know more about properties of protein drugs such as stability, and sensitivity to radiation etc.

We chose to contact Jon Scott, Medical Projects and Technology Team lead for ESA's Space Medicine Office, because he works for the European Space Agency (ESA), has a background in sport and exercise science and did his PhD in about bone metabolism. We thought that it might be very interesting for us to speak with him because bone loss is a serious concern for astronauts due to microgravity in outer space.

We contacted Lonnie Grove, PhD, Postdoc at University of Copenhagen and Department of Orthopedic Surgery, University of California San Diego because of her background as medical doctor and her interest and experience with space and astronaut health. We wanted to speak with Lonnie about what happens to the human body in outer space from a broad perspective.

We contacted Karen McDonald because she (among other things) work with production of protein drugs for astronauts at CUBES (Center for the Utilization of Biological Engineering in Space) . We believed that we had a lot to learn from her and that she might have some interesting inputs about which proteins to produce with our system.

We contacted Eva Horn Møller because she works as Senior Drug Product specialist at Zealand Pharma and we therefore knew that she would have some insight into and experience with production of drugs. Furthermore, she works in the industry and we thought it would be very nice to get some feedback from the industry.

We contacted Jørgen Sauer because he has a background in chemistry and has experience with protein production. We were very interested in getting some feedback on the technical (protein-production) part of our project.

We contacted Michael Hecht, who is a professor at Princeton University, at the European Meetup. He made some interesting suggestions for our system regarding how to handle protein folding.

Integration

Virginia Wotring: The input we got from Virginia made us rethink which space related health issues we wanted to address with our system. Virginia made us consider proteins that affected the immune system and thereby were targeting another type of health problems that we originally hadn't considered. We integrated her suggestion by investigating whether immune regulators would be possible to produce with our protein printer. Virginia also suggested producing G-CSF (granulocyte colony stimulating factor) as a way to address the high radiation levels in outer space. Afterwards we have considered this thoroughly and we have ended up with this as one of the final target proteins for our system!

Jon Scott: Jon Scott encouraged us to try to produce some protein drugs that could be combined with exercise and that would prevent bone loss. This made us readjust our project and try to figure which proteins would be good for preventing bone loss.

Karen McDonald: Karen McDonald suggested producing bone anabolic hormones such as Parathyroid Hormone (PTH) to address bone loss experienced by astronauts. She also suggested producing anti-cancer drugs. This input led us to consider PTH as a possible target protein given that it was interesting from a medical point of view. We further did some analysis of the proteins and we have ended up with PTH being one of our target proteins, which we believe could be produced with our system!

Eva Horn Møller: Eva Horn Møller emphasized the importance of patience safety when producing drugs. Therefore, she said that it was very important that we made a thouroug characterization of if anything was co-secreted through the injectisome. In order to be completely sure, she suggested us to use E. coli disabled of LPS (lipopolysaccharide, which is a molecule that is very toxic to humans) for protein production – and this is definitely something we intend to make sure to disable and which we have incorporated into our project!

Jørgen Sauer: Jørgen Sauer pointed out that we should try to keep the pH in the protein buffer as close to blood pH as possible because this would result less pain when injecting the proteins into the veins. We hadn't thought about this before, but we definitely intend to do it now. Furthermore Jørgen Sauer also pointed out the importance of being aware of the LPS content. This further confirmed our decision made about using E. coli disabled of LPS for protein production. Jørgen Sauer further believed that Human Growth Hormone would be a very good candidate for production with our system.

Based on the experts input (listed above), we made our list of useful proteins that we theoretically should be able to produce with our Protein Printer system (below).

Proteins of interest for space exploration

IgG stan

IgG stan is a collection of IgG antibodies from human donors. The IgG stan is therefore a collection of antibodies with different specificity. IgG is very useful for presenting antigens to the immune system and can therefore function as a booster of the activity of the immune system of the host. Data shows that the immune system is dysregulated under microgravity which might result in an altered immune function [1]. Therefore, it might be interesting to produce proteins that help improve the activity of the immune system. We were pointed in this direction when speaking with Virginia Wotring. Having IgG stan onboard would help boost the immune system of the astronauts.

Immune modulators

As mentioned above the immune system is believed to be dysregulated during microgravity [1]. Therefore, other proteins that affect the immune system could be interesting to produce with our system, in order to reduce this dysregulation. This could both be cytokines, interleukines and other immune regulatory proteins (signal molecules). We were also pointed in this direction by Virginia Wotring. Being able to produce these immune regulatory proteins might make it possible to relieve the different immune system abnormalities that is experienced by astronauts such as allergy, eczema and hypersensitivity.

Granulocyte Colony Stimulating Factor (G-CSF)

Radiation in space is markedly higher and a part of acute radiation syndrome is hematopoietic syndrome. This syndrome is characterized by a reduction in the number of neutrophils. Neutrophils are an important part of the innate immune system and as the number of neutrophils decrease the probability of infections increase. G-CSF promote the differentiation of neutrophils and can therefore be a possible countermeasure to low neutrophil cell count [2][3]. We were also pointed in this direction by Virginia Wotring. Having G-CSF onboard might potentially help the astronauts recover after high levels of radiations.

Insulin like growth factor 1 (IGF-1)

IGF has previously been addressed as a potential therapeutic protein to prevent muscle atrophy [4]. The idea being that IGF-1 acts directly on the target tissue promoting growth. We had thought about producing IGF-1 ourself and discussed this with Jon Scott who thought that it might be very interesting and useful for long term missions to combine these anabolic proteins with exercise in space. Being able to produce IGF-1 onboard would potentially help the astronauts with maintaining muscle mass during space exploration.

Growth hormone (GH)

Growth hormone has both long term and short term effects. The short-term effects are diabetogenic while the long-term effects promotes tissue growth especially muscle and bone. The long-term effects of GH are mediated by IGF-1[5][6]. Therefore both IGF-1 and GH are very closely linked, and whether to use one instead of the other depends on the therapeutical context. We thought about producing GH ourself but were unsure. When speaking with Jon Scott we learned that it might be very interesting to combine anabolic proteins with exercise in space and therefore we chose to consider GH as one of our target proteins. Being able to produce GH onboard might potentially help the astronauts maintain their muscle and bone mass during long term missions.

Parathyroid hormone (PTH)

Karen McDonald suggested we could look into producing Parathyroid Hormone, because it is a bone anabolic hormone and some astronauts suffer from bone loss during microgravity conditions. Parathyroid hormone is produced in parathyroid gland and is secreted in response to low blood calcium concentration. PTH activate the osteoclasts and thereby promote bone resorption and release of calcium and phosphate into the circulation. PTH also promotes secretion of phosphate in the kidney, thereby elevating the concentration of free calcium ions. Despite this PTH also has anabolic effects on bone and is currently used as a osteoporosis drug [7] which makes PTH a very interesting protein for us to produce. Being able to produce PTH onboard might prevent bone loss in astronauts.

Antibody/ single domain antibody

Almost all the experts we have talked to suggested us to produce antibodies, because antibodies are useful for many different things. Our main idea as it is right now it to produce antibodies for detection of different molecules, this could be used both for differential diagnosis of astronauts but also for research purposes and help in a lot of different research purposes in space for instance enlightening what happens to the human body during microgravity. We have looked into which alternatives there is to normal antibodies and found that there exists a single domain antibody that might be easier to produce with our system and are as specific as the human one [8]. Being able to produce single domain antibody on space mission might be useful for research purposes and diagnosis. This would expand the toolbox that the astronauts could bring with them on missions to foreign desolated places. Some types of cancer medicine is based on antibodies [9], therefore being able to produce antibodies might also in the long term lead the way for production of cancer medicine in space.

Requirements for production with our Protein Printer system

Our Protein Printer allows for extremely pure protein to be obtained. However, the system has limitations with regards to the complexity of the proteins that can be produced. The optimal protein to produce with our system should therefore be as simple as possible, able to fold by itself, have a minimum number of post-translational modifications (PTM) and be relatively stable.

Limitations of our system:

1) Folding of the proteins: Once the protein of interest is secreted across the membrane it needs to be able to fold more or less by itself in the conditions of the buffer. This means that smaller and simpler proteins are better to produce with our system given that they are better at folding on their own. How big this folding challenge will be will become clearer when we have data on production of protein with our system. For now we have spoken with Michael Hecht at the European iGEM meetup who suggested we tried to produce his fusion proteins that were better at self-folding. If they work well we could try using his technique for protein production with our system and our protein of interest.

2) Posttranslational modifications: As we produce our proteins using E. coli we are not able to produce proteins with all kinds of posttranslational modifications (PTM). For instance, it is not possible to produce glycosylated proteins in E. coli. PTM is more characteristic for eukaryotic systems, and this fact further complicates protein production, since some PTMs, such as glycosylations, cannot be performed in prokaryotic cells.

Based on the limits of our Protein Printer we have looked at size and PTM's of the proteins we found biologically interesting and wish to produce with our system. This we have done in order to get an idea of the feasibility of producing each protein with our system.

No matter which protein we choose to produce, we will need to optimize the conditions for production taking the specific protein into account. How important the theoretical limitations we present here are, will be further unveiled upon future investigation and experiments.

Conclusion

Based on our research and discussion with expert we propose to use our system for production of IGF-1, PTH, GH, G-CSF and camelid antibody during long lasting space missions. First of all this decision is based on our interviews and discussions with experts. Based on their opinions we have further considered how easy the different proteins would be to produce with our system taking into consideration posttranslational modifications (PTM), protein size and the literature. We have therefore analyzed potential target proteins from several perspectives, especially considering 1) is this protein interesting from a medical point of view? 2) Is this proteins feasible to produce with our system? It has been very important for development of our project to have the possibility to speak with experts such as Virginia Wotring, Karen McDonald, Jon Scott, Jørgen Sauer and Eva Horn Møller and we have very much incorporated their inputs into the very core of our project.

References

[1] B. E. Crucian et al., "Immune system dysregulation during spaceflight: Potential countermeasures for deep space exploration missions," Front. Immunol., vol. 9, no. JUN, pp. 1–21, 2018.
[2] E. Seedhouse, Space Radiation and Astronaut Safety. .
[3] M. P. Mac Manus, D. McCormick, A. Trimble, and W. P. Abram, "Value of granulocyte colony stimulating factor in radiotherapy induced neutropenia: Clinical and laboratory studies," Eur. J. Cancer, vol. 31, no. 3, pp. 302–307, Jan. 1995.
[4] Y.-H. Song, J. L. Song, P. Delafontaine, and M. P. Godard, "The therapeutic potential of IGF-I in skeletal muscle repair.," Trends Endocrinol. Metab., vol. 24, no. 6, pp. 310–9, Jun. 2013.
[5] C. S. Leach, N. M. Cintron, and J. M. Krauhs, "Metabolic changes observed in astronauts," J.Clin.Pharmacol., vol. 31, no. 0091–2700 (Print), pp. 921–927, 1991.
[6] W. Boron and E. Boulpaep, Medical Physiology. 2008.
[7] D. Aslan et al., "Mechanisms for the bone anabolic effect of parathyroid hormone treatment in humans," Scand. J. Clin. Lab. Invest., vol. 72, no. 1, pp. 14–22, Feb. 2012.
[8] M. M. Harmsen and H. J. De Haard, "Properties, production, and applications of camelid single-domain antibody fragments.," Appl. Microbiol. Biotechnol., vol. 77, no. 1, pp. 13–22, Nov. 2007.
[9] A. M. Scott, J. D. Wolchok, and L. J. Old, "Antibody therapy of cancer," Nat. Rev. Cancer, vol. 12, no. 4, pp. 278–287, 2012.