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Synthesising proteins from non-natural building blocks?

Synthetic biology is an emerging discipline that is frequently referred to as the art of molecular engineering. Dr. Birgit Wiltschi from the University of Freiburg has been awarded funding from the Baden-Württemberg Ministry of Science, Research and the Arts under the “Biotechnology and Medical Technology Idea Competition” for a highly ambitious project: she wants to learn how proteins can be modified using non-natural building blocks that will enable the engineered proteins to specifically target receptors on cancer cells, amongst other things. What is the current state of technology? How accurately are biochemists able to construct molecular machines from individual components?

Cancer cells divide more frequently than healthy cells. One of the reasons for this is the higher sensitivity of cancer cells to growth factors such as the epidermal growth factor (EGF) that play an important role in the regulation of cell growth, proliferation and differentiation. The molecule binds to the EGF receptor on the surface of cancer cells and induces a complex signalling cascade that leads to the activation of genes that control cell division activities. Many types of tumours have a high density of EGF receptors on their surface and it is therefore easy to induce cell division. State-of-the-art tumour therapies tend to focus on the targeted and effective inhibition of EGF receptors. And this is exactly what Dr. Birgit Wiltschi from Prof. Dr. Wilfried Weber’s Synthetic Biology workgroup at the Centre for Biological Signalling Studies (BIOSS) in Freiburg intends to do using synthetic biology methods. “Our goal is to create a new class of EGF receptor inhibitors,” said the biochemist. “However, the project is quite risky and we do not know at present whether our approach will lead to the intended objective.”

Control of the building-block pool

Left: Model of an EGF molecule with synthetic tryptophane building blocks (blue). Right: model of a natural tryptophane molecule, including two tryptophane variants into which a nitrogen atom was artificially inserted. © Dr. Birgit Wiltschi

The project, which will be funded for the next nine months under the “Biotechnology and Medical Technology Idea Competition” of the Baden-Württemberg Ministry of Science, Research and the Arts, is still in its early days. Wiltschi and her research group plan to modify the EGF molecule in such a way as to block the function of the EGF receptor on the surface of cancer cells and make cancer cells insensitive to a division signal. Wiltschi’s team will use synthetic biology methods rather than genetic manipulation. Genetic modifications of the coding DNA sequence often lead to protein products that no longer function properly, and this is something the researchers would prefer to avoid. In the case of EGF, such modifications might prevent the peptide molecule from binding to its receptor. Wiltschi and her team plan to target a later step in the production of cellular EGF – namely the point in time at which the amino acids are assembled into a polypeptide. This enables the researchers to use amino acids that are normally not used by the cells. Non-natural building blocks of this kind usually only differ slightly from the structure of natural building blocks; in many cases, the modifications only involve the exchange of a few atoms. But they are atoms that possess the properties the researchers are looking for.

Electron microscope image of E. coli bacteria © Rocky Mountain Laboratories, NIAID, NIH

In order to achieve their goal, Wiltschi and her team are using an approach that has been known since the 1950s, but has only gained importance for the production of synthetic proteins in the last few years. The researchers produce EGF in E. coli cells that are unable to produce the handful of amino acids that would be required for them to carry out protein biosynthesis on their own. “This gives us control over the type of amino acid the bacteria use in their proteins,” said Wiltschi explaining that the researchers are not only able to use amino acids that are normally required for the synthesis of proteins, but can also add synthetically modified amino acids to the culture medium. Since the synthetic and natural building blocks only have minimal structural differences, the three-dimensional structure of the modified proteins remains largely unaltered. However, some of the non-natural building blocks have extraordinary chemical characteristics, which are able to alter the biological function of the synthetic protein variants, potentially in such a way that EGF binds to its receptor and interferes with, rather than activates, its function.

A Trojan Horse or pipe dreams?

Left: Model of a GFP molecule with synthetic proline building blocks (green). Right: model of a natural proline molecule, including a proline variant into which a fluorine atom was inserted. © Dr. Birgit Wiltschi

Wiltschi and her team have already been able to show with other proteins that their approach works in principle. For instance, they were able to replace all the proline residues in green fluorescent protein (GFP) by using minimally modified synthetic proline derivatives, in which a proton is exchanged for a fluorine atom. The substitution of a proton with a fluorine atom led to a GFP protein that folded much more quickly and efficiently into its correct spatial structure. “The structure of the proteins remains the same, but the modified proline molecules alter the folding characteristics of the entire molecule,” said Wiltschi. “This is the fascination – and the potential – of the technology that is being used.”

The researchers now hope to use the aforementioned principle to manipulate the chemical characteristics of EGF molecules. If everything goes to plan, the EGF receptor will not be able to differentiate between modified and natural EGF. Nevertheless, it is likely that the receptor will be blocked, thereby blocking the signalling cascade in the cancer cells and the uncontrolled division of cancer cells. Another possibility would be to insert amino acids that have a toxic effect, which would turn EGF into a kind of Trojan horse that will dock to cancer cells and destroy them. “But it remains to be seen whether all this will work,” said Wiltschi going on to add “at the moment it is still a pipe dream”.

The field of synthetic biology is still very young. Until now, researchers have tested selected methods on model systems and have only been able to show that the methods can be applied. Little is known about the molecular and cellular mechanisms that enable the integration of amino acid analogues into proteins. “Where in the cell do the artificial variants migrate? What exactly happens to them there? Are they part of the cellular metabolism and if so, which enzymes are involved? Wiltschi pointed out that an understanding of the underlying mechanisms is a prerequisite for being able to control specific cellular processes using synthetic biology and use them for specific objectives. There is still a huge amount of basic research to do before the potential of this technology can be exploited in the field of cancer research.

Further information:

Dr. Birgit Wiltschi
Head of Research Group
Institute of Biology II
bioss - Centre for Biological Signalling Studies
University of Freiburg
Engesserstrasse 4b
79108 Freiburg
Tel: +49 (0)761/203-97656
E-mail: birgit.wiltschi(at)biologie.uni-freiburg.de

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/synthesising-proteins-from-non-natural-building-blocks