Within the Human Protein Atlas project, scientists are using ÄKTA systems to purify up to 180 protein samples per week. They wanted to share their knowledge about high-throughput protein purification with readers of GE’s blog.
Hanna, can you tell us a bit about this project?
The Human Protein Atlas, financed by the Knut and Alice Wallenberg Foundation, has been established to systematically study the building blocks of life; proteins. The project uses antibodies to study the localization of proteins in human tissues and cells. The results are freely available in a public database (proteinatlas.org) used by researchers all over the world. Every day several scientific articles are published where antibodies from this project are used.
And your group is responsible for the generation and purification of all these antibodies?
Exactly! In our labs at KTH we start from human RNA pools and produce recombinant expression clones by cDNA synthesis, cloning, and plasmid purification. These clones produce what we call Protein Epitope Signature Tags (PrESTs), a selected part of the target protein that has low homology to other human proteins. We divide each produced PrEST into two aliquots. The PrESTs are used as antigens; we send one aliquot of each for immunization to produce antisera. We couple the PrESTs from the second aliquot to NHS-activated resin in prepacked HiTrap columns, and these columns are used on the ÄKTA systems to purify the specific antisera acquired from the immunization.
Let´s go out to the lab, and meet research engineers Anne-Sophie Svensson and Annelie Cajander. Anne-Sophie, one of your responsibilities is to couple the produced antigens to HiTrap columns?
Yes. We have a high-throughput system, where 6 × 8 HiTrap columns can be coupled at the same time. During one day, our lab has the capacity to couple 96 columns. The coupling is optimized and is usually very straightforward. After coupling the columns can be used for several years. The oldest column in the lab is from the start of the project, in 2003, and is still working!
After coupling the columns are stored in the fridge. It takes about four to five months to receive the sera against the PrESTs. After pre-purification to remove anti-tag antibodies, sera are purified on the ÄKTA system using the coupled HiTrap columns.
Annelie, can you tell us a bit about the setup of your ÄKTA systems?
As you can see, our facility has 16 ÄKTA systems, that each can run four samples. This setup is very good for the high-throughput manner that this whole project operates in. On a typical day the ÄKTA systems are started in the morning. It takes approximately one hour for one person to start six machines, so we usually work in a team to get them all started in the morning. Thereafter, the machine manages itself during the day, and another hour is spent in the afternoon to take care of the samples. We really appreciate that we can work on other things while the machines are running themselves.
And then, after the purification?
We use the UNICORN software to analyze our fractions. I especially like the automatic absorbance measurements of all the fractions, which makes it easy for UNICORN to calculate the protein concentration, and to let us know which fractions to save. In general, I appreciate that the ÄKTA system together with UNICORN is so robust and easy to handle – everyone in our lab can learn it quickly, and it does everything from preparing the columns to running and all the needed washing steps. When running as many as 180 antibody purifications per week we rely on simplicity, reliability, and robustness.
About the interviewer, researchers, and program
Frida Henningson Johnson is communications officer within the Human Protein Atlas. With a PhD in biochemistry and postdoctoral experience in genetics and immunology, she earned a degree in journalism and started working within science communications. She has worked as a freelance science journalist, and within different projects as scientific communicator, writing everything from reports to press releases and social media posts.
Annelie Cajander has an MSc in chemical engineering with a focus on biomolecular engineering, from KTH. She is a research engineer in the Human Protein Atlas (HPA) project since 2008.
Anne-Sophie Svensson Anne-Sophie Svensson has an MSc in biotechnology (engineering) from KTH and started in the HPA project in January, 2013. She is also a research engineer.
Hanna Tegel is site director of the Human Protein Atlas project at the AlbaNova site of KTH. She has been involved in the project since 2003, when the project started. In parallel with her responsibilities within the Human Protein Atlas Hanna has completed a PhD at the Royal Institute of Technology, Stockholm.
The Human Protein Atlas project, funded by the Knut and Alice Wallenberg Foundation, has been set up to allow for a systematic exploration of the human proteome using antibody-based proteomics. This is accomplished by combining high-throughput generation of affinity-purified antibodies with protein profiling in a multitude of tissues and cells assembled in tissue microarrays. Confocal microscopy analysis using human cell lines is performed for more detailed protein localization. The program hosts the Human Protein Atlas portal with expression profiles of human genes and proteins in a multitude of tissues and cells. The main project sites are located at AlbaNova and SciLifeLab, KTH - Royal Institute of Technology, Stockholm, Sweden, and the Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.