There is a beautiful lesson to be learned from the Gossamer Condor[1]where the team of Paul MacCready succeeded where others have failed: to keep a, by man-powered aircraft in the air long enough to draw the infinity sign between two markers one half mile apart, starting and ending the course at least three meters above the ground. They did so on the 23rdof August, 1977.
Paul MacCready sought out to do “do more with less”, however the lesson I learned from this engineering achievement was: “fail fast to succeed faster”. Where other big companies, like Boeing failed, they succeeded. Paul MacCready’s team used cheap and simple materials and techniques so they could build 2 or 3 different configurations a day. They could sample what works and, more importantly, what does not work in a rapid way. Thus, when I start a lecture on making proteins, people look at first confused when I ask them if they have heard of the Kramer prize and its first winner.
So, what does it all have to with making and finding proteins? The process to produce recombinant proteins by use of living cells, currently the state-of-the art especially for the pharmaceutical industry[2], is a lengthy one. For some recombinantly produced antibodies there could be over 1000 different steps. At each of the steps the protein is exposed to different environments, which may interfere with its natural state.
In nature proteins are important, because within the cell and the body they provide structure, perform chemical reactions and pass on information. For example, collagen in connective tissue, α-amylase breaks down long-chain carbohydrates during digestion, or dopamine receptors in the brain. Within cells, many processes are put in place to make the right proteins at the right time, while folding them correctly into their native and active 3D-structures. However, by interfering with these natural processes and tricking cells to make more of the protein we would like to have, for example recombinantly produced human insulin[3], cells may not fold the protein of interest correctly. Misfolded proteins are not active, can induce adverse effects if administered as drugs, and if misfolded protein stick together they form insoluble complexes called aggregates, with no functionality and even greater adverse effects.
Therefore, when we start making small changes to proteins to see if they have an improved functionality compared to its original design, we need to test for many parameters, but functionality and protein stability are crucial. Before we make changes to our proteins, which may have the potential to become safe and functional drugs, we need to make sure our processes are designed well and that our means to evaluate the protein is sound. In the current landscape of protein preparation, the use of cells (i.e. biotechnology) is employed the most. However, one can argue that it takes long to make some protein with many different variations in cells: from weeks to months. How can we fail “faster to succeed faster” to evaluate more proteins?
One disruptive technology which has started to show its promise in an industrial setting[4]is the use of the cellular machinery that makes the proteins without its envelope. Proteins are made by other proteins, enzymes, who transcribe DNA into RNA and translate RNA into an amino acid sequence, a strand of the building blocks of a protein, that then folds into its 3D-structure. This technology is referred to as in-vitro transcription translation (IVTT) or cell free protein synthesis (CFPS)[5]. In short, a cell is cracked open like an egg, and its content is split in a soluble fraction and an in-soluble fraction. Part of the soluble fraction contains all the enzymes needed to make proteins, this part is isolated, often concentrated and then mixed with amino-acids, enzymes to provide energy, and other components needed to synthesize protein. RNA induces the process of protein synthesis, which is often transcribe from DNA within the mixture. Protein production in such an open system is short. Depending on the lysate used the expression can be between 1 – 16 hours, while the process is not designed to keep the cells alive and dividing at a set rate, but on protein synthesis.
Another powerful feature is that many different cell lysates can be used, for example rabbit reticulocyte, E. coli, mammalian cells, such as CHO or HeLa cells, L. tarentolae,tobacco plants, and many others.
The promise of recombinant protein preparation from GMO animals failed in the past; one main reason being the turn-over rate between preparation, purification and testing was very long. One can argue that the turn-over rates of cells are too long as well. In a market where the life-cycles of pharmaceutical products are getting shorter, while the overall volume is growing, more safe and well-designed protein-based drugs are needed. In this landscape CFPS has the potential to act as a valuable tool in the same way mylar foil was crucial for the Gossamer Condor’s success.
Marco G. Casteleijn © 2018
Publish first on BrighOwl blog: https://brightowl.pro/blogs/2018/06/finding-the-best-proteins/
[1]https://en.wikipedia.org/wiki/MacCready_Gossamer_Condor#cite_note-1
[2]Casteleijn M.G. and Richardson D. (2014) Engineering Cells and Proteins – creating pharmaceuticals. European Pharmaceutical Review, 19(4): 12-19
[3]https://www.dnalc.org/view/15255-Producing-human-insulin-using-recombinant-DNA-Walter-Gilbert.html
[4]https://en.wikipedia.org/wiki/Sutro_Biopharma
[5]Marco G. Casteleijn, Arto Urtti, Sanjay Sarkhel. 2013. Expression without boundaries: Cell-free protein synthesis in pharmaceutical research. Int J Pharmaceut, 440 (1): 39-47
Marco Casteleijn 