Spinach Experiments on a Large Scale at Testa Center

Rubisco is an important plant enzyme that absorbs carbon dioxide from the atmosphere. Marvin Seibert’s research team uses leaf spinach to study the protein at the molecular level. The new Testa Center in Uppsala can conduct experiments on a large scale.

Spinach experiments
Marvin Seibert hopes to increase knowledge of how carbon dioxide is fixed in plants. In the foreground: Ekansh Sharma, a Master’s studentPhotograph: Mikael Wallerstedt

Testa Center was inaugurated in August 2018 at GE Healthcare in Boländerna. The facility can be used by both companies and researchers. At Uppsala University Marvin Seibert and his research team were the first to conduct experiments here.

They are associated with the Department of Cell and Molecular Biology and had beam time for neutron scattering experiments in the United States and Germany. Acquiring enough crystallized protein required experiments on a much larger scale than was possible at Uppsala Biomedical Centre (BMC).

“For neutron crystallography you need protein crystals a thousand times larger than for X-ray crystallography,” Marvin Seibert says. “Now that we have run our first experiments at Testa Center, we have more than five grams of protein from two weeks of work, while we produced 100 milligrams at a time in our laboratory.”

The technology is essentially the same as they use at BMC, namely protein chromatography.

“The machines we use here are simply much bigger versions of what we have in our lab. For our process it worked quite well to scale up by about a factor of 40, thanks to the help we received from GE Healthcare’s engineers at Testa Center.”

Before entering the laboratory, we have to don goggles and lab coats. This afternoon the lab rooms are quite empty, but they usually are very busy. After only four months the facility already is operating at two-thirds of full capacity, says Testa Center Director Jesper Hedberg.

“The whole concept – coming here and doing it yourself – is very new to many and will take time to sink in. But both companies and researchers have been enthusiastic. We have had over 1,000 visitors, of which half come from GE and half from outside.”

As director of the facility, Hedberg works hard at disseminating information about Testa Center. “Our mission is making the technology accessible for all who need it.”

To extract rubisco, spinach and a fluid are blended.
PHOTO: MIKAEL WALLERSTEDT

The research team takes a seat at the lab benches and begins to unpack the raw material vital in their research: Leaf spinach, harvested when the slender leaves have just emerged. They contain highly soluble proteins, including rubisco, the enzyme that absorbs carbon dioxide and converts it into energy-rich sugar molecules.

“Rubisco is responsible for almost all carbon dioxide fixation, which is the other side of the carbon equation, making it of fundamental scientific interest. We also hope that a better understanding of this process could reduce the need for fertilizer,” says Seibert.

Initially team members used spinach they grew themselves. Research colleague Marie Wrande has a farm and cultivated the spinach being used in the lab. But as the project grew, the team switched to buying spinach at regular grocery stores.

The leaf spinach is mixed with a liquid into a “smoothie” using a regular blender. Protein can then be extracted from this mixture. This takes place in an exclusion chromatography column, a large tube attached to a machine. Ekansh Sharma, a Master’s student, shows how this is done. He did much of the work last summer along with researcher Dirk Hasse.

The protein is converted into crystals that can be used in experiments in which the chemical reaction is monitored. The technique, called neutron crystallography, will be available at European Spallation Source (ESS), the large facility under construction in Lund. Until it is completed, the research team has to go to facilities in Tennessee in the U.S. and Munich, Germany.

“The technology is phenomenal because we can see the hydrogen and protons in the reaction mechanism,” says Seibert, noting the need for more knowledge.

“I am amazed at how much research we have on how carbon dioxide is released in various processes. But regarding its removal from the atmosphere, the reaction mechanism is literally unknown, simply because we do not know…”

This process cannot be followed with traditional X-ray crystallography because it does not show the position of the protons. It is not even certain this can be done with the new technology, Seibert explains.

“It is a challenge because the protein is so large. But our data from the first experiments looks promising, so we hope we can soon repudiate the claim that it is impossible. We are very close to succeeding in this.”

At Testa Center the research team can produce 40 times as much material in a day or week compared with the lab at Uppsala Biomedical Centre. Ekansh Sharma and Anna Munke check the result of the experiment on the computer monitor. PHOTO: MIKAEL WALLERSTEDT

Crystals are concentrated protein, and they are very fragile. For that reason researchers took the experiments to Munich by car to avoid the risk of damage during shipment by air. When travelling to Tennessee, they flew to Chicago, and then Marvin Seibert spent a day driving to Tennessee. Even so one of two really good crystals was destroyed.

“Of the proteins we most recently extracted, the first batch has already been sent to Tennessee, so we can crystallize it on site. This is a high-risk experiment, and we want to be as well prepared as possible. Testa Center helps us with that,” says Seibert.

Facts

Testa Center is a facility partly funded by Vinnova, Sweden’s innovation agency, at GE Healthcare in Uppsala. The laboratory can be used to scale up various biological processes. Uppsala University and GE Healthcare have entered into a collaboration agreement that offers the University’s researchers the opportunity to test biological processes on a larger scale and students experience in industrial processes.

Neutron crystallography can be carried out at large research facilities abroad. Marvin Seibert’s research team has used the technology at Spallation Neutron Source at Oak Ridge National Laboratory in Tennessee and Research Neutron Source Heinz Maier-Leibnitz in Munich, Germany.

Rubisco is:

  • The predominant link between the atmosphere and biosphere. It captures CO2 out of the air and integrates it into biomass. 
  • The worlds most abundant enzyme.
  • Present in almost all photosynthetic organisms, both on land and at sea.
  • Extremely well conserved over billions of years of evolution. 
  • A very large protein consisting of 16 subunits. 
  • Relatively slow. It catalyses only about 1-10 reactions per second. That’s why there’s so much of it.
  • Healthy to eat. The protein contains all amino acids in a distribution that’s almost perfect for human nutrition. This includes the essential amino acids which we need to obtain from food because our bodies cannot make them.